Methods of preparing and using single chain anti-tumor antibodies

ABSTRACT

This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

This application claims priority of U.S. Ser. No. 60/330,396, filed 17Oct. 2001; Int'l App'l No. PCT/US01/32565, Filed 18 Oct. 2001; and U.S.Ser. No. 10/097,558, Filed 8 Mar. 2002, the content of which areincorporated by reference here into this application.

This application was supported in part by Department Of Energy Grant No.DE-FG-02-93ER61658, National Institutes of Health Grant No. CA61017 andNational Cancer Institute Grant No. NCICA 89936. Accordingly, the UnitedStates Government may have certain rights in this invention.

Throughout this invention, various references are referred to.Disclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

This invention relates to a method for preparation of single chainantibodies, and is of particular applicability to preparation of singlechain Fv antibody fragments (scFv) where the antigen to which theantibody binds is difficult to purify.

ScFv are antibody constructs comprising the variable regions of theheavy and light chains of an antibody as a single chain Fv fragment.ScFv technology utilizes molecular biology methods to reduce antibodiesto the minimal-required-unit of heavy and light chain variable regionstethered by a peptide linker which can be designed with versatile sidechains for radioconjugation.

Procedures for making scFv are known in the art. These proceduresgenerally involve amplification of gene regions encoding the variableregions of the antibodies, assembly of an scFv genetic sequence andexpression of the scFv genetic sequence in host cells. The host cellsare screened using a target antigen to identify those cells which bindto the antigen, ands thus which express a functional scFv of the desiredspecificity. While this procedure works well in many cases, it requiresthe isolation of the antigen for use as a screening tool. In some cases,however, particularly in the case of membrane bound receptor molecules,this isolation may be difficult, or the conformation of the isolatedantigen may be so different that it fails to present the same epitopesfor binding. In these cases, the conventional techniques for developmentof an scFv are either unworkable or very difficult.

SUMMARY OF THE INVENTION

This invention provides a method for identifying cells expressing atarget single chain antibody (scFv) directed against a target antigenfrom a collection of cells that includes cells that do not express thetarget scFv, comprising the step of combining the collection of cellswith an anti-idiotype directed to an antibody specific for the targetantigen and detecting interaction, if any, of the anti-idiotype with thecells, wherein the occurrence of an interaction identifies the cell asone which expresses the target scFv.

This invention also provides a method for inducing proliferation in apopulation of T cells comprising the steps of (a) introducing to the Tcells an expressable gene sequence encoding a scFv coupled to atransmembrane and signaling domain; and (b) exposing the T cells towhich the chimeric scFv has been introduced to an anti-idiotype directedto an antibody specific for a target antigen to which the scFv isdirected under conditions such that the anti-idiotype will bind to scFvon the surface of cells expressing the chimeric scFv thereby inducingproliferation of the T cells.

This invention further provides a method for treating cancer in apatient suffering from cancer expressing an antigenic marker comprisingthe steps of removing lymphocytes from the patient, introducing to thelymphocytes an expressable gene sequence encoding a chimeric scFvcoupled to a transmembrane and signaling domain; exposing thelymphocytes to which the chimeric scFv has been introduced to ananti-idiotype directed to an antibody specific for a target antigen towhich the scFv is directed under conditions such that the anti-idiotypewill bind to scFv on the surface of cells expressing the chimeric scFvand any necessary co stimulatory molecules to induce proliferation ofthe lymphocytes; and returning the expanded population of lymphocytes tothe patient.

In addition, this invention provides a method for treating cancer in apatient suffering from cancer expressing an antigenic marker comprisingthe steps of introducing to human cell lines an expressable genesequence encoding a chimeric scFv coupled to a transmembrane andsignaling domain (including zeta chain); exposing the lymphocytes towhich the chimeric scFv has been introduced to an anti-idiotype directedto an antibody specific for a target antigen to which the scFv isdirected under conditions such that the anti-idiotype will bind to scFvon the surface of cells expressing the chimeric scFv and any necessaryco stimulatory molecules to immunoselect and stimulate clones with highdensity of scFv expression and efficient tumor cytotoxicity to produce agene-modified cell line, and returning the expanded population ofgene-modified cell line to the patient.

This invention also provides a method for enhancing in vivo survival andanti-tumor activity of infused lymphocytes gene-modified withscFv-Chimeric immune receptors by intravenous injection of anti-idiotypeantibody. This invention further provides compositions containing scFv,scFv fusion, cells identified, induced T cell population, alone or incombination thereof.

Finally, this invention provides various uses of the above methods andcompositions.

DETAILED DESCRIPTION OF THE FIGURES

First Series of Experiments

FIG. 1. Inhibition of 8H9 by anti-idiotype 2E9 by FACS analysis. 1A:Staining of LAN-1 neuroblastoma cells with 5 ug/ml of 8H9 (shaded peak)was not inhibited at low concentration of 2E9 (2 ug/ml, black solidline), but almost completely at concentration of 10 ug/ml (dotted line)superimposable with the negative antibody control (grey solid line). 1B:Staining of LAN-1 neuroblastoma cells with 5 ug/ml of 3F8 (anti-GD2,shaded peak) was not inhibited by any concentrations (2 ug/ml, blacksolid line, or 200 ug/ml, dotted line) of 2E9; negative antibody controlthin solid line. 1C: Staining of HTB-82 rhabdomyosarcoma cells with 5ug/ml of 8H9 (grey peak) was not inhibited at low concentration (2ug/ml, grey solid line), but completely at 10 ug/ml of 2E9 (black solidline) superimposable with negative antibody control (black peak).

FIG. 2. SDS-PAGE (lanes a and b) and Western blot (c and d) of ch8H9.H=heavy chain of 8H9, L=light chain of 8H9, arrow points to ch8H9, thefusion protein between 8H9 scFv and the human 1-CH2-CH3 domain. With2-mercaptothanol: lanes a, b and c. Native gel: lane d. SDS-PAGE wasstained with Comassie Blue; western blot with 2E9 anti-idiotypicantibody.

FIG. 3. FACS analysis of ch8H9 and 8H9 staining of HTB82rhabdomyosarcoma and LAN-1 neuroblastoma cells. Mean immunofluorescenceincreased with concentrations of ch8H9 and 8H9, reaching a plateauaround antibody concentration of 3-5 ug/ml. Left Y-axis is meanfluorescence for native 8H9, and the right Y-axis depicts meanfluorescence for ch8H9. Stronger fluorescence for native 8H9 reflects astronger second antibody.

FIG. 4. ch8H9 in antibody-dependent cell-mediated cytotoxicity. ADCC wasmeasured by ⁵¹Cr release as described in Materials and Methods. Percentspecific release is depicted as mean +/− SEM. Target cell line wasrhabdomyosarcoma HTB-82. Control antibody was 3F8 which binds poorly toHTB-82.

FIG. 5. Immunoscintigraphy of human tumors using ¹²⁵I-labeled ch8H9.Mice xenografted with human LAN-1 neuroblastoma received retroorbitalinjections of 25 uCi of ¹²⁵I-labeled antibody. 24 h, 48 h and 7 daysafter injection, the animals were anesthesized and imaged with a gammacamera.

FIG. 6. Blood clearance of ¹²⁵I-labeled ch8H9 and ¹²⁵I-native 8H9. Micexenografted with human LAN-1 neuroblastoma received retroorbitalinjections of ¹²⁵I-labeled antibody. Percent injected dose/gm of serialblood samples were plotted over time.

Second Series of Experiments

FIG. 7. Anti-idiotype affinity enrichment of producer lines. Producerlines were stained with anti-idiotypic MoAb 2E9 before (shaded peak, 7Aand 7B), and after first (dotted line peak, 7A) and second (thick solidline, 7A) affinity purification, and after first (dotted line, 7B) andsecond (solid line 7B) subcloning, showing improved scFv expression. ByFACS the indicator line K562 showed improved scFv expression after first(dotted line, 7C) and second (thick solid line, 7C) affinitypurification of the producer line, and subsequent first (dotted line,7C) and second (thick solid line, 7D) subcloning of the producer line,when compared to unpurified producer lines (shaded peaks, 7C and 7D),consistent with improvement in gene transduction efficiency. The thinsolid line curves in each figure represents nonproducer line (7A and 7B)or uninfected K562 (7C and 7D).

FIG. 8. In vitro expansion of 8H9-scFv-CD28-ζ gene-modified primaryhuman lymphocytes depends on stimulation with anti-idiotypic antibody.Clonal expansion was expressed as fold expansion of initial viablelymphocyte number. IL-2 (100 U/ml [FIG. 8A], 50 U/ml [FIG. 8B] and 20U/ml [FIG. 8C]) was added after retroviral infection and was presentthroughout the entire in vitro culture period, in the presence (solidcircles) or absence (open circles) of solid-phase anti-idiotypicantibody. Viable cell count was performed using trypan blue assay.

FIG. 9. In vitro expansion of 8H9-scFv-CD28-ζ gene-modified primaryhuman lymphocytes from 4 patients with stage 4 neuroblastoma (FIG. 9A)and 4 samples from 2 normal volunteers (FIG. 9B). Clonal expansion wasexpressed as fold expansion of initial viable lymphocyte number beforein vitro culture. IL-2 (100 U/ml) and anti-idiotype antibodies werepresent as described in Materials and Methods. 8H9-scFv-CD28-ζgene-modified lymphocytes underwent continual clonal expansion (103 to108), and survived 150-200 days in vitro, with a double time of ˜5-10days.

FIG. 10. Kinetics of clonal dominance by scFv+ cells and itsrelationship to 8H9scFv gene copy number and 8H9scFv transcript. Percentof lymphocytes positive for surface scFv was monitored by flow cytometryusing anti-idiotype antibody (FIG. 10A); it rapidly increased to near100% by 3 weeks of culture. ScFv gene copy number (PCR, open circles,broken line, FIG. 10B) and scFv transcript (RT-PCR, solid diamonds,solid line, FIG. 10B) also increased with time, reaching their plateauby 10 weeks in culture.

FIG. 11. Cytotoxicity against tumor cell lines: 8H9-scFv-CD28-ζgene-modified lymphocytes (solid circles) from day 56 of culture wereassayed by 51Cr release assay in the presence or absence of MoAb 8H9 (50ug/ml final concentration) as an antigen blocking agent (open circles).Control lymphocytes from the same donor but not gene-modified, werecultured under the same conditions as the gene-modified cells, andtested in cytotoxcity assays in the presence (open diamonds) or absence(solid diamonds) of MoAb 8H9. 11A: NMB-7 neuroblastoma. 11B: LAN-1neuroblastoma. 11C: HTB-82 rhabdomyosarcoma. 11D: Daudi lymphoma.

FIG. 12. Winn assay. suppression of rhabdomyosarcoma tumor growth inSCID mice. Human rhabdomyosarcoma HTB-82 was strongly reactive with 8H9,but not with 5F11 (anti-GD2) antibodies. Experimental groups: HTB-82 wasmixed with 8H9-scFv-CD28-ζ gene-modified human lymphocytes at 3 ratios:1:0.5 (open circle, n=5), 1:1 (solid triangle, n=5), 1:10 (open square,n=10). Control groups: no T-cell (open triangles, n=5), 5F11scFv-CD28-ζmodified lymphocytes at 1:0.5 ratio (solid circles, n=5). Tumor size wascalculated as product of two perpendicular diameters A×B (mean±sem) andplotted over time.

FIG. 13. Suppression of established rhabdomyosarcoma tumor growth inSCID mice. Experimental group: 8H9-scFv-CD28-ζ gene-modified humanlymphocytes+ip 2E9 [rat anti-8H9 anti-idiotype MoAb] (solid circles).Control groups: no cells (open triangles), 5F11scFv-CD28-ζ modifiedlymphocytes+1G8 [rat anti-5F11 anti-idiotype MoAb] (solid squares), and8H9-scFv-CD28-ζ gene-modified human lymphocytes+ip A1G4 [irrelevant ratclass-matched MoAb] (open circles). Relative tumor size was calculatedas % of initial tumor size (Ax B, mean±sem, n=9-10) and plotted overtime.

Third Series of the Experiments

FIG. 14. Transduction of HSV1-tk into primary human T-cells HSV1-tk is atherapeutic gene, a marker gene, as well as a suicide gene. In order toexamine the migration of genetically altered antigen-specific Tlymphocytes to tumors after adoptive transfer in vivo, we exploited thecapacity of transduced T cells expressing HSV-TK to selectivelyphosphorylate and trap in cells and incorporate into DNA radiolabeledthymidine analog 2′-fluoro-2′deoxy-1-D-arabinofuransyl-5-iodo-uracil.

FIG. 15. I131-FIAU Absorbed Dose to Lymphocyte Cell Nuclei. Based on theforgoing dosimetry model and as presented graphically in this figure,the lymphocyte nucleus absorbed dose was calculated as a function ofactivity concentration in the medium and the accumulation ratio. Tostudy the effect on T-cell function, [131I]-labeled FIAU was incubatedwith HSV1-tk transduced T cells at 11 Ci/ml at 37° C. for 40 to 120 minin increasing activity concentrations of [131I]-FIAU from 1.1 to 56μCi/ml, washed and transferred to fresh ([131I]-FIAU-free) medium for 72hr, and then used in a 51Cr-release immune cytotoxicity assay (loweffector:target cell ratio=5). There was no demonstrable diminution inimmune function up to an absorbed dose (at the reference time of 60 hr)of 1,200 cGy. At greater doses (>1,900 cGy), there was a dose-dependentdecrease in immune function.

FIG. 16. Structure of DOTA-DPhe1-Tyr3-octreotide (DOTATOC). Radioactivegallium labeled somatostatin analogue DOTA-DPhe1-Tyr3-octreotide(DOTATOC) for positron emission tomography imaging. Radionuclide labeledsomatostatin analogues selectively target somatostatin receptor(SSTR)-expressing tumors as a basis for diagnosis and treatment of thesetumors. Recently, a DOTA-functionalized somatostatin analogue, DOTATOChas been developed. This compound has been shown to be superior to theother somatostatin analogues as indicated by its uniquely hightumor-to-nontumor tissue ratio. DOTATOC can be labeled with a variety ofradiometals including gallium radioisotopes. Gallium-66 is a positronemitting radionuclide (T1/2=9.5 hr; β+=56%) that can be produced incarrier free form by a low-beam energy cyclotron.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for making a single chainantibody (scFv) directed against a specific, but not necessarilyisolated antigen. The invention further provides a method foridentifying cells expressing the target scFv directed against theantigen, and a method for enriching cell populations expressing thetarget scFv, and for promoting proliferation and expansion of suchpopulations. The invention makes use of an anti-idiotype antibody whichis directed to an antibody specific for the antigen. Cells expressingthe scFv are recognized by the anti-idiotype, thus allowing theirselection. Furthermore, where the scFV is a chimeric immune receptor,which includes a signaling domain in addition to the scFv, theanti-idiotype ligates to these receptors and stimulates proliferation ofscFV expressing T-cells.

The generic invention is illustrated with reference to a specificantibody, designated 8H9, of which the antigen is gp58. 8H9 is a murineIgG1 monoclonal antibody specific for a novel antigen on the cellsurface of a wide spectrum of human solid tumors, but not on normaltissues. In accordance with the invention, scFv directed against gp58antibody was prepared using an anti-idiotype directed against theanti-8H9 monoclonal antibody.

In a first aspect, the present invention provides a method foridentifying cells expressing a target single chain antibody (scFv)directed against a target antigen from a collection of cells thatincludes cells that do not express the target scFv. In a second aspect,the present invention provides a method for making an scFv. In each ofthese aspects of the invention, an anti-idiotype directed against anantibody that is itself directed against the target antigen is utilized.

As used in the specification and claims of this application, the term“directed against” refers to the binding specificity of an antibody. Anantibody which is directed against a particular antigen is one which wasdeveloped and/or selected by a procedure which involves immunization ofan animal with the antigen and/or testing of the antibody for bindingwith the antigen. The antibody may associate with one or a plurality ofepitopes of the target antigen, and may be polyclonal or monoclonal. Theterm “directed against” does not exclude the possibility ofcross-reactivity with other antigens, although antibodies withsubstantial specificity for the particular target antigen are preferred.

Antibodies directed against the target antigen may be prepared usingconventional techniques, including techniques which do not requireisolation or specific knowledge of the antigen. In general, an antigenor a sample for which an antibody is to be developed is administered toan organism to stimulate an immune response. For example, cancer tissuesamples against which it would be desirable to have an antibody can beadministered to mice. Monoclonal antibodies can be developed by fusionof splenic lymphocytes from such immunized mice to myeloma cells toproduce a hybridoma. Selection of hybridoma's producing monoclonalantibodies of the desired specificity is carried out in a routine mannerby testing for the ability to bind to the original target tissue.

Using this general technique, Applicants have isolated a monoclonalantibody designated 8H9. As described in Modak et al., Cancer Res. 61:4048-4054 (2001), monoclonal antibody 8H9 is a murine IgG1 hybridomaderived from the fusion of mouse myeloma SP2/0 cells and spleniclymphocytes from BALB/c mice immunized with human neuroblastoma. Byimmunohistochemistry, 8H9 was highly reactive with human brain tumors,childhood sarcomas, and neuroblastomas, and less so withadenocarcinomas. Among primary brain tumors, 15 of 17 glioblastomas, 3of 4 mixed gliomas, 4 of 11 oligodendrogliomas, 6 of 8 astrocytomas, 2of 2 meningiomas, 3 of 3 schwannomas, 2 of 2 medulloblastomas, 1 of 1neurofibroma, 1 of 2 neuronoglial tumors, 2 of 3 ependymomas, and 1 of 1pineoblastoma tested positive. Among sarcomas, 21 of 21Ewing's/primitive neuroectodermal tumor, 28 of 29 rhabdomyosarcomas, 28of 29 osteosarcomas, 35 of 37 desmoplastic small round cell tumors, 2 of3 synovial sarcomas, 4 of 4 leiomyosarcomas, 1 of 1 malignant fibroushistiocytoma, and 2 of 2 undifferentiated sarcomas tested positive with8H9. Eighty-seven of 90 neuroblastomas, 12 of 16 melanomas, 3 of 4hepatoblastomas, 7 of 8 Wilms' tumors, 3 of 3 rhabdoid tumors, and 12 of27 adenocarcinomas also tested positive. In contrast, 8H9 wasnonreactive with normal human tissues including bone marrow, colon,stomach, heart, lung, muscle, thyroid, testes, pancreas, and human brain(frontal lobe, cerebellum, pons, and spinal cord). Reactivity withnormal cynomolgus monkey tissue was restricted similarly. Indirectimmunofluorescence localized the antigen recognized by 8H9 to the cellmembrane.

These characteristics of the antigen recognized by mAB 8H9(“8H9-antigen”) made it a strong potential candidate as a therapeutictarget. 8H9 immunoprecipitated a Mr 58,000 band after N-glycanasetreatment, most likely a protein with a heterogeneous degree ofglycosylation. However, the antigen is proteinase sensitive and is noteasily modulated off the cell surface. Thus, preparation and isolationof an scFv which could be used for targeting cells expressing the8H9-antigen required the development of a different approach.

In accordance with the method of the present invention, the antibodydirected against the target antigen is used to create an anti-idiotypeantibody. The anti-idiotype antibody can be produced in any species,including human (preferably using in vitro immunization), although mouseand rat will most commonly be immunized because of the convenience ofworking with such animals in the laboratory. The anti-idiotype ispreferably prepared as a monoclonal antibody to make it easier toproduce or purify. Once made, the anti-iditoype can be used to screenscFv libraries from any species to identify scFv antibodies directedagainst the target of the original antibody used to create theanti-idiotype. Thus, as illustrated in the examples below, a ratanti-mouse idiotype was used to screen a human cDNA scFv library.

The procedures for creating scFv libraries are known in the art.Generally, the procedures involve amplification of the variable regionsof nucleic acids encoding an antibody, commonly from a hybridomaproducing an antibody of interest. Generic primers associated with theconstant regions of such antibodies are available commercially. Theamplified fragments are then further amplified with primers selected tointroduce appropriate restriction sites for introduction of the scFvinto an expression vector, phage, or fusion protein. Cells producing thescFv are screened and an scFv with the desired selectivity isidentified.

The scFv which is identified can be used in any of numerousapplications. For example, the scFv can be labeled, for example using aradiolabel, a colored or chromogenic label or a fluorescent label, andused for diagnostic testing of tissue samples to detect the presence ofa tumor-associated target antigen (such as the 8H9-antigen) or otherdiagnostic antigenic marker. Tissue samples are exposed to the labeledscFv for a period of time to allow specific interaction if the targetantigen is present. The sample is washed to remove non-specificallybound materials, and binding of the label to the cells is indicative ofthe presence of the marker. A similar approach may be used forhistological mapping of the location of antigenic markers in tissuesections and samples. The scFv may also be used as one component insandwich type assays, such as ELISA, and may be used as affinity probesfor capture and purification of the target antigen.

The scFv may be used as a targeting moiety to direct chemotherapy agentsto specific cell types. The DNA encoding the scFv may also be combinedto produce a genetic sequence encoding a fusion protein. Examples oftypes of fusion proteins which may be created include scFv-cytokine (Shuet al., Proc. Nat'l Acad. Sci. (USA) 90: 7995-7999 (1993),scFv-streptavidin (Kipriyanov et al., Human Antibody Hybridomas 6:93-101 (1995); WO 97/34634), scFv-enzyme (Michael et al., Immunotech. 2:47-57 (1996)), scFv-toxin (Wickstrand et al., Cancer Res. 55: 3140-48(1995)), bispecific scFv (diabodies) (Alt et al., FEBS Letters 454:90-94 (1999)), bi-specific chelating scFv (De Nardo et al., Clin.,Cancer Res. 5: 3213s-3218s (1999)), scFv-Ig (Shu et al., supra),tetravalent scFv (Alt et al., supra, Santos et al., Clin., Cancer Res.5: 3118s-3123s (1999)), and scFv-retargeted T cells (Eshar et al., Proc.Nat'l Acad. Sci (USA) 90: 720-724 (1993)).

In one specific embodiment of the invention, the scFv is coupled in afusion protein to T-cell signaling and transmembrane domains. Expressionof such fusion proteins in T cells leads to presentation of the scFv onthe surface of the T cell. Proliferation and expansion of such T cellscan be induced by exposing the T cells to which the chimeric scFv hasbeen introduced to an anti-idiotype directed to an antibody specific fora target antigen to which the scFv is directed under conditions suchthat the anti-idiotype will bind to scFv on the surface of cellsexpressing the chimeric scFv. Furthermore, such T cells will be targetedin vivo to cells which express the target antigen. Thus, the presentinvention also provides a method for treating a disease conditioncharacterized by the presence of cells expressing a characteristicsurface antigen, comprising the steps of developing an scFv to thetarget antigen and forming a genetic sequence encoding a fusion proteinof the scFv with T cell signaling and transmembrane domains; recoveringlymphocytic cells from the patient and transforming the cells ex vivo sothat they express the fusion protein, stimulating proliferation andexpansion of the cells by exposing the cells ex vivo to ananti-idiotype, and returning the cells to the patient.

This invention also provides compositions comprising the scFv, scFvfusion, and cells expressing scFv, respectively. This invention providesa pharmaceutical composition comprising scFv alone, scFv fusion alone,cells expressing scFv alone, or any combination thereof. This inventionfurther provides a pharmaceutical composition comprising scFv alone,scFv fusion alone, cells expressing scFv alone, or any combinationthereof and a pharmaceutically acceptable carrier. For the purposes ofthis invention, “pharmaceutically acceptable carriers” means any of thestandard pharmaceutical carriers. Examples of suitable carriers are wellknown in the art and may include, but are not limited to, any of thestandard pharmaceutical carriers such as a phosphate buffered salinesolution and various wetting agents. Other carriers may includeadditives used in tablets, granules and capsules, etc. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gum, glycols or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well-known conventional methods.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

From these and the foregoing description, it can be seen that theinvention provides:

A method for identifying cells expressing a target scFv directed againsta target antigen from a collection of cells that includes cells that donot express the target scFv, comprising the step of combining thecollection of cells with an anti-idiotype directed to an antibodyspecific for the target antigen and detecting interaction, if any, ofthe anti-idiotype with the cells, wherein the occurrence of aninteraction identifies the cell as one which expresses the target scFv.The cells identified by the above method. A composition comprising saidcells.

A method for making a scFv directed against an antigen, wherein theselection of clones is made based upon interaction of those clones withan appropriate anti-idiotype, and heretofore inaccessible scFv so made.

The single chain antibody made by the above method and a compositioncomprising said scFv.

A method for selecting cell lines that package and produce high titersof retroviral particles that carry the scFv gene for transfection intocells. The cells include but are not limited to human lymphocytes.

The selected cell lines from the above method and composition comprisingthe same.

A method for inducing proliferation in a population of T cellscomprising the steps of (a) introducing to the T cells an expressablegene sequence encoding a chimeric scFv coupled to a transmembrane andsignaling domain; and (b) exposing the T cells to which the chimericscFv has been introduced to an anti-idiotype directed to an antibodyspecific for a target antigen to which the scFv is directed underconditions such that the anti-idiotype will bind to scFv on the surfaceof cells expressing the chimeric scFv thereby inducing proliferation ofthe T cells.

The population of T cells induced by the above method and compositioncomprising the same.

A method for tagging cells to facilitate sorting or enrichment,comprising the steps of expressing a scFv in the cell and capturing ortagging the cells using anti-idiotype.

A method for isolating an antigen comprising the steps of preparing anantibody to the antigen, preparing an anti-idiotype directed to theantibody, using the anti-idiotype to select a scFv, targeting theantigen from a scFv library, and using the selected scFv as an affinityprobe to capture antigen, preferably with the scFv immobilized on asolid support.

The isolated antigen by the above method and a composition comprisingthe same.

A method for treating cancer in a patient suffering from cancerexpressing an antigenic marker comprising the steps of removinglymphocytes from the patient, introducing to the lymphocytes anexpressable gene sequence encoding a chimeric scFv coupled to atransmembrane and signaling domain; exposing the lymphocytes to whichthe chimeric scFv has been introduced to an anti-idiotype directed to anantibody specific for a target antigen to which the scFv is directedunder conditions such that the anti-idiotype will bind to scFv on thesurface of cells expressing the chimeric scFv and any necessary costimulatory molecules to induce proliferation of the lymphocytes; andreturning the expanded population of lymphocytes to the patient. Theantigenic marker includes but is not limited to gp58 and GD2.

A method for treating cancer in a patient suffering from cancerexpressing an antigenic marker comprising the steps of introducing tohuman cell lines an expressable gene sequence encoding a chimeric singlechain antibody (scFv) coupled to a transmembrane and signaling domain(including zeta chain); exposing the lymphocytes to which the chimericscFv has been introduced to an anti-idiotype directed to an antibodyspecific for a target antigen to which the scFv is directed underconditions such that the anti-idiotype will bind to scFv on the surfaceof cells expressing the chimeric scFv and any necessary co stimulatorymolecules to immunoselect and stimulate clones with high density of scFvexpression and efficient tumor cytotoxicity to produce a gene-modifiedcell line, and returning the expanded population of gene-modified cellline to the patient. The antigenic marker includes but is not limited toGD2 and gp58. The human cell line includes but is not limited to NK92,natural killer, helper, and cytotoxic cell line. The gene-modified cellline produced by the above method. A composition comprising thegene-modified cell line.

A method for enhancing in vivo survival and anti-tumor activity ofinfused lymphocytes gene-modified with scFv-Chimeric immune receptors byintravenous injection of anti-idiotype antibody.

EXPERIMENTAL DETAILS

First Series Of Experiments

Anti-Idiotypic Antibody as the Surrogate Antigen for Cloning scFv andIts Fusion Proteins

ScFv is a versatile building block for novel targeting constructs.However, a reliable screening and binding assay is often the limitingstep for antigens that are difficult to clone or purify. We demonstratethat anti-idiotypic antibodies can be used as surrogate antigens forcloning scFv and their fusion proteins. 8H9 is a murine IgG1 monoclonalantibody specific for a novel antigen expressed on the cell surface of awide spectrum of human solid tumors but not in normal tissues (CancerRes 61:4048,2001) Rat anti-8H9-idiotypic hybridomas (clones 2E9, 1E12and 1F11) were produced by somatic cell fusion between rat lymphocytesand mouse SP2/0 myeloma. In direct binding assays (ELISA) they werespecific for the 8H9 idiotope. Using 2E9 as the surrogate antigen,8H9-scFv was cloned from hybridoma cDNA by phage display. 8H9scFv wasthen fused to human-γ1-CH2-CH3 cDNA for transduction into CHO and NSOcells. High expressors of mouse scFv-human Fc chimeric antibody wereselected. The secreted homodimer reacted specifically withantigen-positive tumor cells by ELISA and by flow cytometry, inhibitableby the anti-idiotypic antibody. The reduced size resulted in a shorterhalf-life in vivo, while achieving comparable tumor to nontumor ratio asthe native antibody 8H9. However, its in vitro activity inantibody-dependent cell-mediated cytotoxicity was modest.

Introduction

Single chain Fv (scFv) has greatly expanded the potential anddevelopment of antibody-based targeted therapies.⁽¹⁻⁴⁾ Using phagedisplay, scFv can now be cloned from cDNA libraries derived fromrodents, immunized volunteers, or patients.⁽⁵⁻⁸⁾ The availability ofhIg-transgenic and transchromosomal mice will allow immunization schemaor pathogens not feasible or safe in humans.

Construction of the scFv is the critical first step in the synthesis ofvarious fusion proteins, including scFv-cytokine,⁽⁹⁾scFv-streptavidin,⁽¹⁰⁾ scFv-enzyme,⁽¹¹⁾ scFv-toxins,⁽¹²⁾ bispecific scFv(diabodies),⁽¹³⁾ bispecific chelating scFv,⁽¹⁴⁾ scFv-Ig,⁽⁹⁾ tetravalentscFv^((13,15)) and scFv-retargeted T-cells.⁽¹⁶⁾ ScFv-Ig constructs mimicnatural IgG molecules in their homodimerization through the Fc region,as well as their ability to activate complement (CMC) and mediateantibody dependent cell-mediated cytotoxicites (ADCC).

The construction of scFv requires a reliable antigen preparation bothfor panning phages and for binding assays. They often become arate-limiting step,⁽¹⁷⁾ particularly for antigens that are difficult toclone or purify. Cell-based phage display,⁽¹⁸⁾ and enzyme linkedimmunosorbent assays (ELISA) when optimized, have been successfullyapplied as alternatives. Subtle differences in the panning step candetermine the success or failure of phage display.⁽¹⁹⁾ For example, areduction in wash pH is needed for scFv directed at ganglioside GD2 inorder to reduce nonspecific adherence of phage particles.⁽¹⁹⁾ Moreover,phage binding assay may require membrane preparations to withstand thevigorous washing procedure.

As antigen mimics of infectious agents and tumor antigens,anti-idiotypic antibodies have promising clinical potentials.⁽²⁰⁻²²⁾They are convenient surrogates when the target antigen is not readilyavailable. The physico-chemical behavior of immunoglobulins as antigensin panning and binding assays is generally known and can be easilystandardized. Hombach et al successfully isolated scFv with specificityfor CD30 utilizing internal image anti-idiotypic antibodies.⁽²³⁾ Werecently described a novel tumor antigen reactive with a murine MoAb8H9.⁽²⁴⁾ Given its lability and glycosylation, this antigen is difficultto purify. Here we describe the use of an anti-idiotypic antibody as asurrogate antigen for cloning a scFv derived from the 8H9 hybridoma cDNAlibrary, and for the selection of chimeric mouse scFv-human Fc fusionconstructs. This provides a proof of principle for isolating antibodiesof same specificity from a non-specific phage display library.

Materials and Methods

Animals

BALB/c mice were purchased from Jackson Laboratories, Bar Harbor, Me.Lou/CN rats were obtained from the National Cancer Institute-FrederickCancer Center (Bethesda, Md.) and maintained in ventilated cages.Experiments were carried out under a protocol approved by theInstitutional Animal Care and Use Committee, and guidelines for theproper and humane use of animals in research were followed.

Cell Lines

Human neuroblastoma cell lines LAN-1 was provided by Dr. Robert Seeger(Children's Hospital of Los Angeles, Los Angeles, Calif.), and NMB7 byDr. Shuen-Kuei Liao (McMaster University, Ontario, Canada). Cell lineswere cultured in 10% defined calf serum (Hyclone, Logan, Utah) in RPMIwith 2 mM L-glutamine, 100 U/ml of penicillin (Sigma-Aldrich, St. Louis,Mo.), 100 ug/ml of streptomycin (Sigma-Aldrich), 5% CO₂ in a 37° C.humidified incubator. Normal human mononuclear cells were prepared fromheparinized bone marrow samples by centrifugation across aFicoll-Hypaque density separation gradient. Human AB serum (GeminiBioproducts, Woodland, Calif.) was used as the source of humancomplement.

Monoclonal Antibodies

Cells were cultured in RPMI 1640 with 10% newborn calf serum (Hyclone,Logan, Utah) supplemented with 2 mM glutamine, 100 U/ml of penicillinand 100 ug/ml of streptomycin (Sigma-Aldrich). 3F8, an IgG3 MoAb raisedin a BALB/c mouse against human neuroblastoma, specifically recognizesthe ganglioside GD2. The BALB/c myeloma proteins MOPC-104E, TEPC-183,MOPC-351, TEPC-15, MOPC-21, UPC-10, MOPC-141, FLOPC-21, and Y5606 werepurchased from Sigma-Aldrich. MoAb R24 (anti-GD3), V1-R24, and K9(anti-GD3) were gifts from Dr. A. Houghton, OKB7 and M195 (anti-CD33)from Dr. D. Scheinberg, and 10-11 (anti-GM2) from Dr. P. Livingston ofMemorial Sloan Kettering Cancer Center, New York; and 528 (EGF-R) fromDr. J. Mendelsohn of M D Anderson, Houston, Tex. 2E6 (rat anti-mouseIgG3) was obtained from hybridomas purchased from American Type CultureCollection [ATCC] (Rockville, Md.). NR-Co-04 was provided by GeneticsInstitute (Cambridge, Mass.). In our laboratory, SF9, 8H9, 3A5, 3E7,1D7, 1A7 were produced against human neuroblastoma; 2C9, 2E10 and 3E6against human breast carcinoma, and 4B6 against glioblastoma multiforme.They were all purified by protein A or protein G (Pharmacia, Piscataway,N.J.) affinity chromatography.

Anti-8H9 Anti-Idiotypic Antibodies

LOU/CN rats were immunized intraperitoneally (ip) with 8H9 (400 μg perrat) complexed with rabbit anti-rat serum (in 0.15 ml), and emulsifiedwith an equal volume (0.15 ml) of Complete Freund's Adjuvant (CFA)(Gibco-BRL, Gaithersburg, Md.). The 8H9-rabbit-IgG complex was preparedby mixing 2 ml (8 mg) of purified 8H9 with 4 ml of a high titer rabbitanti-rat precipitating serum (Jackson Immunoresearch Laboratories, WestGrove, Pa.). After incubation at 4° C. for 3 hours, the precipitate wasisolated by centrifugation at 2500 rpm for 10 minutes, and resuspendedin PBS. Three months after primary immunization, the rats were boostedip with the same antigen in CFA. One month later, a 400 μg boost of8H9-rabbit-anti-mouse complex was injected intravenously. Three daysafterwards, the rat spleen was removed aseptically, and purifiedlymphocytes were hybridized with SP2/0-Ag14 (ATCC). Clones selection wasbased on specific binding to 8H9 and not to control antibody 5F9, amurine IgG1. Repeated subcloning using limiting dilution was done.Isotypes of the rat monoclonal antibodies were determined by MonoclonalTyping Kit (Sigma-Aldrich). Rat anti-idiotypic antibody clones (2E9,1E12, 1F11 ) were chosen and produced by high density miniPERMbioreactor (Unisyn technologies, Hopkinton, Mass.), and purified byprotein G affinity chromatography (Hitrap G, Pharmacia). The IgGfraction was eluted with pH 2.7 glycine-HCl buffer and neutralized with1 M Tris buffer pH 9. After dialysis in PBS at 4° C. for 18 hours, thepurified antibody was filtered through a 0.2 um millipore filter(Millipore, Bedford, Mass.), and stored frozen at −70° C. Purity wasdetermined by SDS-PAGE electrophoresis using 7.5% acrylamide gel. 2E9was chosen from among the three anti-idiotypic antibodies because of itshigh titer.

The “standard” ELISA to detect rat anti-idiotypic antibodies (Ab2) wasas follows: Purified 8H9, or irrelevant IgG1 myeloma, were diluted to 5ug/ml in PBS and 50 μl per well was added to 96-well flat-bottomedpolyvinylchloride (PVC) microtiter plates and incubated for 1 hour at37° C. Rows with no antigen were used for background subtraction. Fillerprotein was 0.5% BSA in PBS and was added at 100 μl per well, andincubated for 30 minutes at 4° C. After washing, 50 μl duplicates ofhybridoma supernatant was added to the antigen-coated wells andincubated for 3 hours at 37° C. The plates were washed and aperoxidase-conjugated mouse anti-rat IgG+IgM (Jackson ImmunoresearchLaboratory) at 100 μl per well was allowed to react for 1 hour at 4° C.The plate was developed using the substrate o-phenylenediamine(Sigma-Aldrich) (0.5 mg/ml) and hydrogen peroxide (0.03%) in 0.1 Mcitrate phosphate buffer at pH 5. After 30 minutes in the dark, thereaction was quenched with 30 μl of 5 N sulfuric acid and read using anELISA plate reader.

Specificity by Direct Binding Assay

Fifty μl per well of purified mouse monoclonal antibodies or myelomaswere coated onto 96-well PVC microtiter plates at 5 ug/ml for 60 minutesat 37° C., aspirated and then blocked with 100 μl of 0.5% BSA fillerprotein per well. After washing and air-drying, the wells were allowedto react with anti-idiotypic antibodies. The rest of the procedure wasidentical to that described in the “standard” assay.

Specificity by Inhibition Assay

To further examine the specificity of these anti-idiotypic antibodies,inhibition of 8H9 immunofluorescent staining of tumor cells byanti-idiotypic antibodies was tested. Purified 8H9 and anti-GD2 MoAb3F8, (all 10 ug/ml in 0.5% BSA) were preincubated with variousconcentrations of anti-idiotypic antibodies for 30 minutes on ice beforereacting with 10⁶ cells of either GD2-positive/8H9 positive LAN-1(neuroblastoma) or GD2-negative/8H9-positive HTB-82 (rhabdomyosarcoma).The cells were then washed twice in PBS with 0.1% sodium azide andreacted with FITC-conjugated rat anti-mouse IgG (Biosource, Burlingame,Calif.) on ice for 30 minutes in the dark. The cells were washed in PBSwith azide, fixed in 1% paraformaldehyde and analyzed by FACScan(Becton-Dickinson, Calif.). The mean fluorescence was calculated and theinhibition curve computed.

Construction of scFv Gene

mRNA was isolated from 8H9 hybridoma cells using Quick Prep Micro mRNAPurification kit (Pharmacia Biotech). 5×10⁶ hybridoma cells cultured inRPMI-1640 medium supplemented 10% calf serum, L-glutamine (2 mmol/L),penicillin (100 u/L) and streptomycin sulphate (100 ug/ml) were pelletedby centrifugation at 800×g and washed once in RNase-free phosphatebuffered saline (pH 7.4). Cells were lysed directly in the extractionbuffer and Poly(A)-RNA was purified by oligo (dT)-cellulose. The mRNAsample was precipitated from the elution buffer using 100 μg glycogen,40 μl of 2M potassium acetate solution and 1 ml of absolute ethanol at−20° C. for 1 hour. The nucleic acid was recovered by centrifugation at10,000×g for 30 min. The sample was evaporated until dry, and dissolvedin 20 μl RNase-free water.

ScFv gene was constructed by recombinant phage display. 5 μl of mRNA wasreverse-transcribed in a total volume of 11 μl reaction mixture and 1 μldithiothreitol (DTT) solution for 1 hour at 37° C. For PCR amplificationof immunoglobulin variable regions, light chain primer mix and the heavychain primer sets (Pharmacia) were added, to generate suitablequantities of the heavy (340 bp) and light (325 bp) chains. Following aninitial 10 min dwell at 95° C., 5U AmpliTaq Gold DNA polymerase (AppliedBiosystems, Foster City, Calif.) was added. The PCR cycles consisted ofa 1 min denaturation step at 94° C., a 2 min annealing step at 55° C.and a 2 min extension step at 72° C. After 30 cycles of amplification,PCR derived fragment was purified by the glassmilk beads (Bio101, Vista,Calif.) and separated by 1.5% agarose gel electrophoresis in TAE buffer,then visualized by ethidium bromide staining. For the assembly andfill-in reaction, both purified heavy chain and light chain fragmentswere added to an appropriate PCR mixture containing a 15 amino acidlinker-primer for 8H9, dNTPs, PCR buffer and Ampli Taq Gold DNApolymerase. PCR reactions were performed at 94° C. for 1 min, followedby a 4 min annealing reaction at 63° C. The heavy and light chain DNA of8H9 were joined by the linker (GGGS)₃ (Pharmacia) into scFv-in a VH-VLorientation after 7 thermocycles. Using an assembled scFv DNA of 8H9 astemplate, a secondary PCR amplification (30 standard PCR cycles) wascarried out using primers containing either Sfi I or Not I restrictionsites. Thus, the Sfi I and Not I restriction sites were introduced tothe 5′ end of heavy chain and the 3′ end of light chain, respectively.Amplified ScFv DNAs were purified by glassmilk beads and digested withSfi I and Not I restriction endonucleases. Digestion with Sfi I wascarried out in NEBuffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mMDithiothreitol, pH 7.9) for 4 hours at 50°C. NotI digestion was carriedout in 100 mM NaCl for 4 hours at 37° C. The purified ScFv of 8H9 wasinserted into the pHEN1 vector (kindly provided by Dr. G. Winter,Medical Research Council Centre, Cambridge, UK) containing Sfi I/Nco Iand Not I restriction sites. Competent E. coli XL 1-Blue cells(Stratagene, La Jolla, Calif.) were transformed with the pHEN1 phagemid.Helper phage M13 KO7 (Pharmacia) was added to rescue the recombinantphagemid.

Enrichment of recombinant phagemid by panning 50 μl of anti-8H9idiotypic antibody 2E9 (50 ug/ml) in PBS was coated on the 96-well PVCmicrotiter plates and incubated at 37° C. for 1 hour. 100 μl of thesupernatant from phage library was added to each well and incubated for2 hours. The plate was washed 10 times with PBS containing 0.05% BSA.Antigen-positive recombinant phage captured by the anti-idiotype MoAb2E9 was eluted with 0.1 M glycine-HCl (pH 2.2 containing 0.1% BSA) andneutralized with 2M Tris solution. This panning procedure was repeatedthree times. The phagemid 8HpHM9F7-1 was chosen for the rest of theexperiments.

ELISA

The selected phage was used to reinfect E. coli XL 1-Blue cells.Colonies were grown in 2xYT medium containing ampicillin (100 ug/ml) and1% glucose at 30° C. until the optical density of 0.5 unit at 600 nm wasobtained. Expression of scFv antibody was induced by changing to themedium containing 100 μM IPTG (Sigma-Aldrich) and incubating at 30° C.overnight. The supernatant was separated by centrifugation. Afterresuspending the pellet in PBS containing 1 mM EDTA and incubating onice for 10 min, the soluble antibody in the periplasmic fraction wascollected by centrifugation. Both supernatant and periplasmic fractionswere added to plates coated with anti-idiotype 2E9. After a 2 hourincubation at 37° C., plates were washed and reacted with anti-MycTagantibody (clone 9E10 from ATCC) for 1 hour at 37° C., and susbeqeuntlywith affinity purified goat anti-mouse antibody (Jackson Immunoresearch)for 1 hour at 37° C. The plates were developed with the substrateo-phenylenediamine (Sigma-Aldrich) as previously described.

Construction of ScFv-Human-1-CH2-CH3 Mouse Human-Chimeric Gene

A single gene encoding scFv8H9 was generated by PCR method usingphagemid 8HpHM9F7-1 as the template. Secondary PCR amplification (30 PCRcycles) was carried out to insert the human IgG1 leader sequence at the5′end of the scFv8H9 DNA plus the restriction sites at the two oppositeends, i.e. Hind III and Not I, at the 5′ end of human IgG1 leader and atthe 3′ end of scFv8H9, respectively. Amplified human IgG1 leader—scFv8H9DNA was purified by glassmilk beads and digested with Hind III and Not Irestriction endonucleases according to manufacturer's instructions. TheHind III—Not I fragment of human IgG1 leader-scFv8H9 cDNA was purifiedon agarose gel and ligated into pLNCS23 vector carrying thehuman-γ1-CH2-CH3 gene (kindly provided by Dr. J. Schlom, National CancerInstitute, NIH, Bethesda, Md.)⁽⁹⁾. Competent E. coli XL 1-Blue cellswere transformed with pLNCS23 containing the scFv phagemid. ThescFv-CH2-CH3 DNA was amplified with appropriate primers and sequencedusing the Automated Nucleotide Sequencing System Model 373 (AppliedBiosystems). The sequences agreed with the cDNA sequences of the lightand heavy chains of 8H9 as well as the human-γ1-CH2-CH3 (GenBank),including the ASN 297 of the CH2 domain. In this construct, Cys220 ofthe genetic hinge was replaced by a proline residue, while Cys226 andCys229 were retained in the functional hinge⁽⁹⁾

Cell Culture and Transfection

CHO cell or NSO myelomas cells (Lonza Biologics PLC, Bershire, UK) werecultured in RPMI 1640 (Gibco-BRL) supplemented with glutamine,penicillin, streptomycin (Sigma-Aldrich) and 10% fetal bovine serum(Gibco-BRL). Using effectene transfection reagent (Qiagen, Valencia,Calif.), recombinant cFv8H9-human-γ1-CH2-CH3 was introduced via thepLNCS23 into CHO cell or NSO myelomas cells. Cells were fed every 3days, and G418 (1 mg/ml; Gibco-BRL) resistant clones were selected.After subcloning by limiting dilution, chimeric antibodies were producedby high density miniPERM bioreactor from Unisyn Technologies using 0.5%ULG-FBS in Hydridoma-SFM (Invitrogen Corporation, Carlsbad, Calif.). Thechimeric antibodies were purified by protein G Pharmacia) affinitychromatography.

SDS-PAGE and Western Blot Analysis

The supernatant, the periplasmic extract and cell extract from thepositive clones were separated by reducing and nonreducing SDS-PAGE. 10%SDS-polyacrylamide slab gel and buffers were prepared according toLaemmli.⁽²⁵⁾ Electrophoresis was performed at 100V for 45 min. Aftercompletion of the run, western blot was carried out as described byTowbin.⁽²⁶⁾ The nitrocellulose membrane was blocked by 5% nonfat milk inTBS solution for 1 hour and incubated with anti-idiotype 2E9 antibodyovernight at 4° C. After incubating with HRP-conjugated goat anti-rat Ig(Fisher Scientific Co., Pittsburgh, Pa.), the signal was detected by ECLsystem (Amersham-Pharmacia Biotech).

Cytotoxicity Assay

Target NMB7 or HTB-82 tumor cells were labeled with Na₂ ⁵¹CrO₄ (AmershamPharmacia) at 100 uCi/10⁶ cells at 37° C. for 1 hour. After the cellswere washed, loosely bound ⁵¹Cr was leaked for 1 hour at 37° C. Afterfurther washing, 5000 target cells/well were admixed with lymphocytes toa final volume of 200 μl/well. Antibody dependent cell-mediatedcytotoxicity (ADCC) was assayed in the presence of increasingconcentrations of chimeric antibody. In complement mediated cytotoxicity(CMC), human serum as source of complement (at 1:40, 1:80, 1:160, 1:320,1:640 dilution) was used instead of lymphocytes. The plates wereincubated at 37° C. for 4 hours. Supernatant was harvested usingharvesting frames (Skatron, Lier, Norway). The released ⁵¹Cr in thesupernatant was counted in a universal gamma-counter (PackardBioscience, Meriden, Conn.). Percentage of specific release wascalculated using the formula 100%×(experimental cpm—background cpm)/(10%SDS releasable cpm—background cpm), where cpm were counts per minute of⁵¹Cr released. Total release was assessed by lysis with 10% SDS(Sigma-Aldrich), and background release was measured in the absence ofcells. The background was usually <30% of total for either NMB7 orHTB-82 cells. Antibody 3F8 was used as the positive control.⁽²⁷⁾

Iodination

MoAb was reacted for 5 min with ¹²⁵I (NEN Life Sciences, Boston, Mass.)and chloramine T (1 mg/ml in 0.3M Phosphate buffer, pH 7.2) at roomtemperature. The reaction was terminated by adding sodium metabisulfite(1 mg/ml in 0.3M Phosphate buffer, pH 7.2) for 2 min. Free iodine wasremoved with A1GX8 resin (BioRad, Richmond, Calif.) saturated with 1%HSA (New York Blood Center Inc., New York, N.Y.) in PBS, pH 7.4.Radioactive peak was collected and radioactivity (mCi/ml) was measuredusing a radioisotope calibrator (Squibb, Princeton, N.J.). Iodineincorporation and specific activities were calculated. Trichloroaceticacid (TCA) (Fisher Scientific) precipitable activity was generally >90%.

In vitro Immunoreactivity of Iodinated Antibody

Immunoreactivity of radioiodine labeled antibody was assayed usingpurified anti-idiotype antibody 2E9 as the antigen. Appropriatedilutions of ¹²⁵I labeled antibodies were added to plates in duplicates,and then transferred to freshly prepared antigen plates after 1 h and 4h of binding at 4° C., respectively. The final binding step was allowedto proceed overnight at 4° C. The total percent radioactivity bound wasa summation of 3 time points for each antibody dilution. For native 8H9,maximum immunoreactivity averaged ˜65%, while 8H9 scFv-Fc (ch8H9)antibody was ˜48%.

Animal Studies

Athymic nude mice (nu/nu) were purchased from NCI, Frederick Md. Theywere xenografted subcutaneously with LAN-1 neuroblastoma cell line(2×10⁶ cells/mouse) suspended in 100 μl of Matrigel (Beckton-DickinsonBioSciences, Bedford, Mass.) on the flank. After 3 weeks, mice bearingtumors of 1-1.5 cm in longest dimension were selected. Animals wereinjected intravenously (retrorbital plexus) with 20 μCi of ¹²⁵I labeledantibody. They were anesthesized with ketamine (Fort Dodge AnimalHealth, Fort Dodge, Pa.) intraperitoneally and imaged at various timeintervals with a gamma camera (ADAC, Milpitas, Calif.) equipped withgrid collimators. Serial blood samples were collected at 5 min, 1, 2, 4,8, 18, 24, 48, 72, 120 h from mice injected with 10-11 uCi ¹²⁵I labeledantibody. Groups of mice were sacrificed at 24 h, 48 h, and 120 h andsamples of blood (cardiac sampling), heart, lung, liver, kidney, spleen,stomach, adrenal, small bowel, large bowel, spine, femur, muscle, skin,brain and tumor were weighed and radioactivity measured by a gammacounter. Results were expressed as percent injected dose per gram.Animal experiments were carried out under an IACUC approved protocol,and institutional guidelines for the proper and humane use of animals inresearch were followed.

Results

Anti-8H9-Idiotypic Antibodies

Rat hybridomas specific for 8H9 and nonreactive with control murine IgG1were selected. After subcloning by limiting dilution, rat antibodieswere produced by bulk culture in roller bottles and purified by proteinG affinity column. By ELISA, 2E9, 1E12, and 1F11, all of rat subclassIgG2a, were specific for 8H9, while nonreactive with a large panel ofpurified monoclonal antibodies (Table 1). In contrast, the antibodies3C2, 4C2 5C7, 7D6 and 8E12 from the same fusions were not specific for8H9. The rest of the experiments in this study was carried out usingantibody 2E9 because of its high titer in vitro. 2E9 specificallyinhibited the binding of 8H9 to LAN-1 neuroblastoma (FIG. 1A) and HTB82rhabdomyosarcoma (FIG. 1B) while control rat IgG1 (A1G4) had no effect(FIG. 1C).

Construction and Expression of 8H9 ScFv

After three rounds of panning of the recombinant phagemid on theanti-idiotypic antibody 2E9, the eluted phage was used to infect E. coliHB2151 cells and scFv expression was induced by IPTG. ScFv fromperiplasmic soluble protein fraction was tested for binding to 2E9 onELISA. Three 8H9 scFv clones when compared with the MoAb 8H9 showedsimilar titers. The clone 8HpHM9F7-1 was selected for subcloning. TheDNA sequence of 8HpHM9F7-1 agreed with those of the 8H9VH and 8H9VL.

The supernatant, periplasmic soluble and cells pellet lysates of8HpHM9F7-1 were separated by nonreducing SDS-PAGE, and analyzed bywestern blotting. A protein band with molecular weight of 31KD was foundin the supernatant, the periplasmic and cell pellet extracts usinganti-MycTag antibody which recognized the sequence GAPVPDPLEPR. (SEQ IDNO. 18) No such band was detected in control cells or 8HpHM9F7-1 cellswithout IPTG treatment.

Construction of Chimeric Mouse scFv-Human Fc

Chimeric clones from CHO and NSO were screened by ELISA binding on 2E9.Clone 1C5 from NSO and clone 1G1 from CHO were chosen for scale-upproduction. By SDS-PAGE and by western blot analysis, a single chain of54 kD under reducing conditions, and a homodimer of 102 kD undernonreducing conditions were found (FIG. 2). Antigen specificity wasdemonstrated by its binding to tumor cells. In FIG. 3, mean fluorescenceplateaued around 3-5 ug/mL of both ch8H9 and 8H9 for both HTB-82rhabdomyosarcoma and LAN-1 neuroblastoma cells, while negative (<10%mean fluorescence) for the control cell line Daudi (data not shown).Cell staining (5 ug/ml of ch8H9) was completely inhibited by 1 ug/ml ofanti-idiotypic antibody 2E9 on FACS analysis (data not shown). DNAsequencing confirmed the presence of 8H9scFv and the CH2-CH3 domain ofhuman Fcγ1.

In Vitro and In Vivo Properties of ch8H9

The ch8H9 antibody mediated ADCC in the presence of human lymphocyteswith a 16% maximum cytotoxicity at 50:1 E:T ratio, significantly higherthan the controls 3F8 or 8H9 (FIG. 4A). However, it was unable tomediate CMC in the presence of human complement (data not shown). Inbiodistribution studies, it localized well to HTB82 and LAN-1 xenografts(FIG. 5). Blood clearance studies showed that chimeric 8H9 (102 kD MW)had T-1/2 of 5.3 h, and T-1/2 of 43 h when compared to averages of 4.5 hand 71 h, respectively, for native 8H9 (160 kD MW), a result of thesmaller molecular size of the construct (FIG. 6). Similarly, althoughthe percent injected dose per gram of the chimeric construct (Table 2)was lower for all tissues (average of 44% at 48 h, and 75% at 120 h),the tumor-non tumor ratios (Table 3) were similar to those of native 8H9(98% at 48 h and 85% at 120 h).

Discussion

We demonstrated that by using rat anti-idiotypic antibody as antigensurrogate, scFv and scFv-fusion proteins can be conveniently produced.As proof of principle we utilized the anti-idiotypic antibody to clonescFv from the murine hybridoma cDNA library. The anti-idiotypic antibodywas then used to select for scFv-Fc chimeric antibodies. Both the scFvand scFv-Fc fusion protein derived by our method were specific for thenatural antigen, comparable to the native antibody 8H9.

While scFv provides the building block for scFv-fusion proteins, it isnot the ideal targeting agent by itself. Being a small protein, itsclearance is rapid. Moreover, it is often retained by the kidney,delivering undesirable side effects if the scFv construct is cytotoxic.Since avidity is a key parameter in tumor targeting in vivo, its biggestlimitation is its uni-valency and often suboptimal affinity for theantigen. By using VH-VL linkers of decreasing length, spontaneousdimeric, trimeric and polymeric scFv have been produced. However, theseoligomers are not bonded by covalent linkage, and may dissociate invivo. An alternative approach is to take advantage of the human Fc,which has the natural ability to homodimerize through disulfide-bonds,thereby allowing the juxtaposition of two binding domains. Fc functionssuch as CMC and ADCC could also be achieved.^((9,28-31))

Unlike standard 2-chain chimeric antibodies, only one polypeptide isneeded for the scFv-Fc chimeric; unbalanced synthesis of heavy and lightchains is not an issue. Larger dimeric fragments are also likely to haveincreased serum-half life compared to scFv and thus improved tumortargeting.^((32,33)) Homodimerization of tumor cell-surface antigens bysoluble antibody may also trigger apoptosis of tumor cells.⁽³⁴⁾ No lessimportant is the availability of validated purification techniques usingprotein A or protein G through their binding to the Fc portion.⁽³¹⁾Tetravalent scFv (monospecific or bispecific) are natural extensions ofthe diabody approach to scFv-Fc fusion strategy,^((13,15)) where asignificant increase in avidity can be achieved. More recently,scFv-streptavidin fusion protein has been produced for pretargetedlymphoma therapy.⁽³⁵⁾ Here scFv-streptavidin forms natural tetramers, towhich biotinyated ligands can bind with high affinity.

Anti-idiotypic antibodies have greatly facilitated clone selection inthe construction of soluble scFv-fusion proteins or cell bound surfacescFv. We have successfully applied similar technology to anti-GD2monoclonal antibodies.⁽³⁶⁾ Being immunoglobulins, their structure,stability, biochemistry, are generally known. Unlike natural antigenswhere each individual system has its unique and difficult to predictproperties. As surrogate antigens, anti-idiotypic antibodies are idealfor standardization and quality control, especially for initial clinicalinvestigations where the nature of the antigen is not fully understood.Potential limitations exist for the anti-idiotype approach. Only thoseanti-ids (Ab2 ) that recognize the antigen-binding site of theimmunizing MoAb can mimic the original antigen. A reliable test for Ab2is its ability to induce an antigen-specific immune response.Alternatively, antigen specificity of the scFv selected by theanti-idiotype must be validated by binding to cells or membranepreparations.

Once validated, the anti-idiotype can be used as antigen surrogate forcloning and assay of other scFv-fusion proteins. Although our scFv-Fcfusion protein ch8H9 mediated ADCC, it could not mediate CMC. Thisfinding differs from previous scFv-Fc fusion proteins.^((9,30,31)) It ispossible that the affinity of the antibody 8H9 may be suboptimal tomediate efficient ADCC/CMC; or that the p58 antigen and tumor lines usedmay not be optimal targets for CMC. Alternatively, poor in vitro Fcfunction may relate to the oligosaccharide structures in the Fcregion.⁽³⁷⁾ In normal IgG, these oligosaccharides are generally ofcomplex biantennary type, with low levels of terminal sialic acid andbisecting N-acetylglucosamine (GlcNAc), the latter being critical forADCC. ADCC function is often inefficient among chimeric antibodiesexpressed in cell lines which lack the enzyme(1,4)-N-acetylglucosaminyltransferase III (GnIII),⁽³⁸⁾ that catalyzesthe formation of bissecting oligosaccharides. This enzyme can betransfected into producer lines to increase the level of bisectingGlcNAc and to increase the ADCC function of secreted chimericantibodies.⁽³⁸⁾ It is also possible that the absence of the CH1 domainin the Fc may modify the accessability of the ASN297 residue toglycosyltransferases in some scFv-Fc constructs such as ours.⁽³⁷⁾ On theother hand, an scFv-Fc that lacks binding to Fc receptor may have lessnonspecific binding to white cells, thereby decreasing blood pooling intargeted therapy. These findings may have implications in scFv-Fcstrategies to improve effector functions.

TABLE 1 Anti-8H9-idiotypic antibodies: Specificity by ELISA 1E12 1F113C2 4C2 5C7 7D6 8E12 2E9 MoAb Class 2a 2a 2b μ μ 1 μ 2a MOPC-315 a − −+++ − − − − − 20.4 1 − − +++ +++ ++ +++ − − 2C9 1 − − +++ +++ +++ +++ ++− 2E10 1 − − +++ − − + − − 3E6 1 − − +++ +++ +++ +++ +++ − 3E7 1 − − +++− − + − − 4B6 1 − − +++ +++ ++ +++ − − 5F9 1 − − +++ +++ +++ +++ + − 8H91 +++ ++ +++ +++ ++ +++ − ++ MOPC-21 1 − − +++ +++ +++ +++ − − UJ 13A 1− − +++ ++ + − − − 3A5 2a − − +++ − − − − − MOPC-1 2a − − +++ + − − − −3F8 3 − − +++ − − − − − FLOPC-21 3 − − +++ ++ − ++ − − NRCO-04 3 − − +++− − − − − R24 3 − − +++ − − − − − TIB114 3 − − +++ + − ++ − − Y5606 3 −− +++ − − − − − 3A7 μ − − + − − − − − 3G6 μ − − +++ − − − − − 5F11 μ −− + − − − − − K9 μ − − +++ − − − − MOPC-104E μ − − +++ − − − − − Note:OD < 0.5 = −, 0.5~1 = +, 1~2 = ++, >2 = +++

TABLE 2 Percent Injected Dose per gram over time in hours Percentinjected dose/gm over time (h) mean +/− se Chimeric Native Organs 24 48120 48 120 Skin 1.4 +/− 0.2 +/− 0.7 +/− 0.2 +/− 1.8 +/− 0.2 0.1 0.0 0.00.2 Heart 1.3 +/− 0.2 +/− 0.9 +/− 0.4 +/− 2.6 +/− 0.2 0.1 0.0 0.2 0.2Lung 2.9 +/− 0.4 +/− 1.9 +/− 0.5 +/− 4.0 +/− 0.4 0.3 0.1 0.3 0.3 Liver1.2 +/− 0.1 +/− 0.8 +/− 0.2 +/− 1.4 +/− 0.1 0.1 0.0 0.2 0.2 Spleen 0.9+/− 0.2 +/− 0.5 +/− 0.2 +/− 1.4 +/− 0.2 0.0 0.1 0.2 0.1 Kidney 1.5 +/−0.1 +/− 0.9 +/− 0.5 +/− 1.9 +/− 0.1 0.1 0.2 0.1 0.1 Adrenal 0.9 +/− 0.1+/− 0.5 +/− 0.5 +/− 1.8 +/− 0.1 0.2 0.2 0.5 0.0 Stomach 1.3 +/− 0.3 +/−0.6 +/− 0.3 +/− 1.3 +/− 0.3 0.1 0.1 0.1 0.3 Small 0.6 +/− 0.1 +/− 0.3+/− 0.2 +/− 0.7 +/− intestine 0.1 0.0 0.1 0.0 0.1 Large 0.6 +/− 0.1 +/−0.3 +/− 0.2 +/− 0.6 +/− intestine 0.1 0.0 0.1 0.1 0.0 Bladder 1.2 +/−0.1 +/− 0.6 +/− 0.4 +/− 1.0 +/− 0.1 0.2 0.2 0.2 0.2 Muscle 0.5 +/− 0.1+/− 0.3 +/− 0.2 +/− 0.5 +/− 0.1 0.0 0.1 0.0 0.1 Femur 0.6 +/− 0.1 +/−0.3 +/− 0.2 +/− 0.8 +/− 0.1 0.0 0.1 0.1 0.0 Spine 0.6 +/− 0.1 +/− 0.4+/− 0.2 +/− 0.8 +/− 0.1 0.0 0.1 0.1 0.1 Tumor 4.0 +/− 0.3 +/− 3.6 +/−2.1 +/− 9.4 +/− 0.3 0.5 0.4 1.3 0.5 Brain 0.2 +/− 0.0 +/− 0.1 +/− 0.1+/− 0.2 +/− 0.0 0.0 0.0 0.0 0.0 Blood 5.3 +/− 0.3 +/− 3.1 +/− 1.2 +/−8.3 +/− 0.3 0.3 0.2 0.7 0.8

TABLE 3 Tumor to normal tissue over time in hours Tumor to normal tissueratio over time (h), mean +/− se Chimeric Native Organs 24 48 120 48 120Skin 3.0 +/− 6.0 +/− 10.7 +/− 5.2 +/− 7.2 +/− 0.3 1.3 1.7 0.7 2.2 Heart3.3 +/− 4.0 +/− 5.6 +/− 3.6 +/− 7.7 +/− 0.7 0.7 0.4 0.3 2.9 Lung 1.6 +/−2.2 +/− 4.5 +/− 2.3 +/− 5.0 +/− 0.4 0.5 0.7 0.3 1.7 Liver 3.5 +/− 5.2+/− 8.7 +/− 6.5 +/− 10.1 +/− 0.5 1.3 1.1 0.4 3.4 Spleen 5.1 +/− 8.1 +/−12.8 +/− 6.7 +/− 15.1 +/− 1.0 1.6 3.4 0.4 5.7 Kidney 2.8 +/− 4.3 +/− 5.9+/− 5.1 +/− 8.9 +/− 0.3 1.1 1.6 1.0 1.1 Adrenal 4.8 +/− 8.7 +/− 10.0 +/−5.8 +/− 11.6 +/− 0.5 2.3 3.2 1.3 1.6 Stomach 3.6 +/− 6.7 +/− 13.8 +/−7.5 +/− 14.5 +/− 0.8 1.3 4.2 1.7 4.3 Small 6.6 +/− 11.8 +/− 16.0 +/−13.3 +/− 21.7 +/− intestine 0.7 2.1 3.7 2.2 6.1 Large 7.1 +/− 12.7 +/−25.9 +/− 15.7 +/− 28.5 +/− intestine 1.0 2.2 7.1 3.4 8.9 Bladder 3.5 +/−14.3 +/− 10.2 +/− 12.4 +/− 12.3 +/− 0.3 9.2 3.3 5.5 5.3 Muscle 7.9 +/−13.6 +/− 21.3 +/− 18.2 +/− 26.8 +/− 0.7 2.4 6.8 1.3 9.6 Femur 6.7 +/−11.8 +/− 20.5 +/− 11.8 +/− 27.9 +/− 1.1 2.4 6.8 1.3 6.5 Spine 6.7 +/−6.8 +/− 14.2 +/− 11.1 +/− 19.6 +/− 0.9 1.9 3.7 1.1 6.2 Tumor 1.0 +/− 1.0+/− 1.0 +/− 1.0 +/− 1.0 +/− 0.0 0.0 0.0 0.0 0.0 Brain 22.7 +/− 40.9 +/−38.7 +/− 44.6 +/− 68.2 +/− 2.9 8.6 10.4 10.4 35.2 Blood 0.8 +/− 1.2 +/−1.8 +/− 1.1 +/− 2.3 +/− 0.1 0.2 0.3 0.1 0.8

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Experimental Details

Chimeric immune receptors (CIR) transduced into lymphocytes link targetrecognition by single chain antibody Fv (scFv) to activation throughCD28/TCRζ signaling. As surrogate antigens, anti-idiotypic antibodiesmay facilitate gene-transduction and clonal expansion of humanlymphocytes for in vivo tumor therapy. The murine monoclonal antibody(MoAb) 8H9 reacts with a novel antigen widely expressed on solid tumors(Cancer Res 61:4048, 2001). A CIR consisting of human CD8-leadersequence, 8H9scFv, CD28 (transmembrane and cytoplasmic domains), andTCR-ζ chain was constructed, ligated into the pMSCVneo vector, and usedto transfect the packaging line GP+envAM12 bearing an amphotropicenvelope. Rat anti-idiotypic MoAb 2E9 (IgG2a) was used to cloneretroviral producer line as well as to expand gene-modified primaryhuman lymphocytes. Sequential enrichments using either affinitychromatography or cell sorting using anti-idiotypic MoAb 2E9significantly improved the percentage of producer clones positive forsurface 8H9-scFv and the efficiency of their supernatant in transducingthe indicator cell line K562. By three weeks of in vitro culture, >95%of transduced primary human lymphocytes were CIR-positive. Upon periodicstimulation with 2E9, these lymphocytes underwent >10⁶ fold expansion by6 months in culture. They mediated antigen-specific non-MHC restrictedcytokine release and tumor cytotoxicity. When admixed with tumor cellsor injected intravenously, they inhibited human xenograft growth in SCIDmice. Anti-idiotypic antibody may provide a useful tool for optimizinggene transduction of CIR fusion constructs into primary humanlymphocytes and their continual expansion in vitro. Adoptive celltherapy using ex vivo expanded tumor-selective T-cells can effectdramatic remissions of virally induced malignancies, a processcritically dependent on clonal frequency, where rapid exponentialexpansion of specific cytolytic T-lymphocytes (CTL) isrequired.(Papadopoulos, 1994 #2140; Heslop, 1997 #4703) T-cellsproliferate when activated (e.g. anti-CD3). However, apoptosis occursunless a costimulatory signal (e.g. anti-CD28) is present.(Daniel, 1997#4714) However, human tumor targets often lack costimulatory molecules(e.g. CD80), or overstimulate inhibitory receptors (e.g. CTL4) such thatthe CD28 pathway is derailed. In addition, many tumors downregulatemajor histocompatibility complex (MHC) molecules to escape engagement bythe T-cell receptor (TCR). Through genetic engineering, chimeric immunereceptors (CIR) linking tumor-selective scFv to T-cell signaltransduction molecules (e.g. TCR-ζ chain and CD28) will activatelymphocytes following tumor recognition, triggering the production ofcytokines and tumor lysis. (Eshhar, 1993 #6028;Stancovski, 1993 #2392;Moritz, 1994 #3399; Wels, 1995 #2452; Hwu, 1993 #2394; Eshhar, 2001#5665; Rossig, 2001 #6088; Ma, 2002 #6437) T-cell can also begenetically engineered to secrete cytotoxic cytokines,(Rosenberg, 1995#2372) toxins, (Yang, 1997 #3395) or to metabolize prodrugs. (Culver,1992 #3478; Wei, 1994 #3466) However, significant technologic gapsremain: (1) Gene transduction into primary human lymphocytes isinefficient, (2) Antigen specific T-cells cannot be easily enriched andexpanded, (3) Optimal T-cell activation may require multiple signals,and (4) Demonstration of anti-tumor effect of these human T-cells inestablished tumor models has been difficult and so far unsuccessful inpatients. (Ma, 2002 #6437) Furthermore, although CIR redirected T-cellscan recycle their lytic activity,(Weijtens, 1996 #3396) a costimulatorysignal, either through CD28 or 4-1BB engagement, may help reduceactivation-induced apoptotic death.(Maus, 2002 #6438) CIR withmultidomains was recently described, where the intracellular domain ofCD28 was ligated to the 5′ end of TCR-ζ chain and introduced into Jurkatcells, with the expected “two signals” when scFv was triggered by tumorcells. (Finney, 1998 #4574) IL-2 production was 20 times more than CIRwith ζ-chain only. Primary mouse CD8+ T lymphocytes expressing thescFv-CD28-ζ receptor secreted Tc1 cytokines, induced T-cellproliferation, and inhibited established tumor growth and metastasis invivo, a process shown to be critically dependent on IFN-γ secretion.(Haynes, 2002 #6477) CD28-mediated cytokine secretion through CIRactivation was recently demonstrated in primary human T-cells. (Krause,1998 #3956; Maher, 2002 #6433)

To monitor scFv gene expression, anti-linker antibody may be useful.However, its efficiency depends on the accessibility of the scFv-linkerportion. Although purified antigens can also be used to monitor scFvexpression, certain classes (complex carbohydrates or unstable antigens)can be difficult to prepare and their chemistry highly variable. Withouta standardized reagent for affinity purification or enrichment of virusproducer cells, as well as monitoring and sorting of transducedlymphocytes, CIR technology remains inefficient. A dicistronic constructconsisting of scFv-CD28-γ and green fluorescent protein (GFP) exploitedthe latter was to monitor gene transduction and to enrich producerlines.(Eshhar, 2001 #5665) Although GFP can validate the gene transferprocess, its added immunogenicity and its safety in clinicalapplications remain uncertain.

Anti-idiotypic antibodies are frequently used as antigen-mimics forinfectious diseases and cancer. Thanavala, 1986 #3290; Wagner, 1997#6019) Internal image rat anti-idiotypic antibodies can be convenientlyproduced against mouse MoAb. Since large scale production of clinicalgrade MoAb is now routine, anti-idiotypic antibodies may be idealsurrogates especially if the antigen is not readily available. Inaddition, the biochemistry of immunoglobulins in positive selection(panning, affinity chromatography, sorting) and binding assays iswell-known and is easy to standardize. Single chain antibody fragmentswith specificity for cell surface antigens were successfully isolated byphage display utilizing internal image anti-idiotypic antibodies.(Hombach, 1998 #6363) We recently described a novel tumor antigenreactive with a murine MoAb 8H9. (Modak, 2001 #3872) The antigen wasdifficult to purify given its lability and glycosylation. Here wedemonstrate that an anti-idiotypic MoAb against 8H9 can be used as asurrogate antigen for cloning CIR into primary human lymphocytes, i.e. aCIR of 8H9scFv, human CD28 and human TCR-ζ chain. While previous studiesshowed that anti-idiotypic antibody can enhance cytotoxicity ofscFv-γR-gene modified murine cytotoxic T-cell line, (Reinhold, 1999#4340) we now show that anti-idiotypic MoAb, besides allowing rapidaffinity enrichment of producer cell line and monitoring of surface scFvexpression, induces clonal expansion of CIR-modified primary humanlymphocytes. Highly cytotoxic lymphocytes can be propagated in vitroundergoing 10⁶ fold expansion over a period of 6 months.

Materials and Methods

Materials Cells were cultured in RPMI 1640 with 10% newborn calf serumHyclone, Logan, Utah) supplemented with 2 mM glutamine, 100 U/ml ofpenicillin and 100 ug/ml of streptomycin. 8H9 murine IgG1 monoclonalantibody directed at gp58 on human solid tumors has been previouslydescribed.(Modak, 2001 #3872) Anti-idiotypic antibodies were producedfrom LOU/C N rats. (Cheung, 1993 #1499) Clones (2E9, 1E12, 1F11) wereselected based on selective binding to 8H9 antibody and not to othermyelomas. After repeated subcloning, 2E9 (rat IgG2a) was chosen for itshigh in vitro antibody production using high density miniPERM bioreactor(Unisyn technologies, Hopkinton, Mass.), and purified by protein Gaffinity chromatography (Hitrap G, Amersham-Pharmacia, Piscataway,N.J.). The IgG fraction was eluted with pH 2.7 glycine-HCl buffer andneutralized with 1 M Tris buffer pH 9. After dialysis in PBS at 4° C.for 18 hours, the purified antibody was filtered through a 0.2 umMillipore filter (Millipore Inc. Bedford Mass.), and stored frozen at−70° C. Purity was determined by SDS-PAGE electrophoresis using 7.5%acrylamide gel. ELISA was used to detect rat anti-idiotypic antibodies(Ab2) as previously described.(Cheung, 1993 #1499) Rat IgG1 anti-5F11anti-idiotypic MoAb 1G8 was similarly produced.

Construction of ScFv Gene scFv was constructed from 8H9 hybridoma cDNAby recombinant phage display (Amersham-Pharmacia). Amplified ScFv DNA,purified by glassmilk beads, restriction digested (Sfi I and Not I), andinserted into the pHEN1 vector (kindly provided by Dr. G. Winter,Medical Research Council Centre, Carmbridge, UK). Competent E. Coli XL-1Blue cells (Stratagene, La Jolla, Calif.) were transformed with thepHEN1 phagemid. Following rescue with the helper phage M13 KO7(Pharmacia), recombinant phagemids were enriched by panning. 50 ul ofanti-8H9 idiotypic antibody 2E9 (50 ug/ml) in PBS were coated on the96-well polyvinyl microtiter plates and incubated at 37° C. for 1 hour.100 ul of the supernatant from phage library were added to each well andincubated for 2 hours. The plate was washed 10 times with PBS containing0.05% BSA. Antigen-positive recombinant phage captured by anti-idiotypicMoAb 2E9 was eluted with 0.1M HCl (pH 2.2 with solid glycine and 0.1%BSA) and neutralized with 2M Tris solution. This panning procedure wasrepeated three times. The phagemid 8HpHM9F7-1 was chosen for the rest ofthe experiments. The appropriate size scFv (31 kD) was demonstrated inthe supernatant, periplasmic and cell extracts by nonreducing SDS-PAGEand western blotting.(Towbin, 1979 #6020) using anti-Myc Tag antibody(clone 9E10 from ATCC, Rockville, Bethesda, Md.).

ELISA The selected phage was used to reinfect E. coli XL-1 Blue cells.Colonies were grown in 2xYT medium containing ampicillin (100 ug/ml) and1% glucose at 30° C. until the optical density at 600 nm of 0.5 wasobtained, and expression of scFv antibody was induced with 100 uM IPTG(Sigma-Aldrich) at 30° C. overnight. Both supernatant and periplasmicfractions were assayed for scFv on anti-idiotype 2E9 coated plates.After incubating 2 hours at 37° C., plates were washed and reacted withanti-MycTag antibody for 1 hour at 37° C. After washing, affinitypurified goat anti-mouse antibody (Jackson Immunoresearch, West Grove,Pa.) was allowed to react for 1 hour at 37° C. and the plates weredeveloped with the substrate o-phenylenediamine (Sigma-Aldrich).

Construction of sc8H9-hCD28_(TM)-hCD28_(cyto)-hTCRζ-pMSCVneo Using theassembled gene sequences, secondary PCR amplifications using syntheticoligodeoxynucleotide primers (see below) were performed. Briefly, a 50μl reaction mixture containing 200 μM of each deoxynucleotidetriphosphate, 0.2 μM of each primer, 2 units of AmpliTag Gold DNApolymerase (Appled Biosystems, Foster City, Calif.), and 50 ng oftemplate DNA was subjected to a 10 min denaturation and activation stepat 95° C., followed by 30 cycles of denaturation (1 min at 95° C.),annealing (2 min at 55° C.), and extension (2 min at 72° C.). This wasfollowed by a final extension for 8 min at 72° C. Each of the amplifiedproducts was purified with Geneclean Kit (Bio 101, Vista, Calif.).

Synthetic Oligodeooxynucleotide Primers for DNA Amplification (1) hCD8aleader - scFv - CD28: Sense Primer (Hpa I - Human CD8a Leader ) 5′ - TTATTA CGA GTT/AAC ATG GCC TTA CCA GTG ACC - 3′ (SEQ ID NO. 1) AntisensePrimer (Xho I - Human CD2B ) 5′ - CTT GGT C/TCGAG TGT CAG GAG CGA TAGGCT GC - 3′ (SEQ ID NO. 2) (2) Bh9scFv: Sense Primer (Cla I - 8H9 heavychain) 5′ - TTA TTA CGA AT/CGATT GCC CAG GTC AAA CTG - 3′ (SEQ ID NO. 3)Antisense Primer (Not I - 8H9 light chain ) 5′ - CTT GGT G/CGGCCGC CTGTTT CAG CTC CAG - 3′ (SEQ ID NO. 4) (3) 5F11scFv: Sense Primer ( Cla I -5F11 heavy chain) 5′ - TTA TTA CGA AT/CGAT TCA GCA GTC AGG ACC - 3′ (SEQID NO. 5) Antisense Primer (Not I - 5F11 light chain) 5′ - CTT GGT G/CGGCC GC CCG TTT TAT TTC CAA CTG - 3′ (SEQ ID NO. 6) (4) hTcR-ζ chain:Sense primer (Bst U I - CD28 end - Xho I - hTCR-ζ [cytoplasmic domain])5′ - CG/C GAC TTA GCA GCC TAT CGC TCC TGg CAC/ TCG AGa AGA GTG AAG TTC -3′ (SEQ ID NO. 7) Antisense Primer (Bg1II - hTCR z) 5′ CTT GGT A/GA TCTTCA GCG AGG GGG CAG GGC - 3′ (SEQ ID NO. 8)

Templates for DNA Amplification and Construction The single geneencoding hCD8a-leader-sc3G6-CD28 was previously described. (Krause, 1998#3956) Its cDNA was generated by PCR using the Hpa I, Xho I fragment ofhCD8a-leader-scFv-CD28 cDNA, and ligated into pMSCVneo vector (Clontech,Palo Alto, Calif.). ScFv-8H9 was amplified from the 8HpHM9F7-1 phagemid,and the excised 8H9 scFv gene swapped into the hCD8a-leader-scFv3G6-CD28cassette of pMSCVneo using the Cla I-Not I restriction enzymes. HumanTCR-ζ chain was amplified from the plasmid pcDNA3.1/VJABLZH (kindlyprovided by Dr. Ira Bergman, University of Pittsburgh, Pa.), and ligateddownstream of CD28 gene, using Xho I and Bgl II restriction sites. Usingthe method supplied by manufacturer (Stratagene), competent E. coli XL-1Blue cells were transformed with the vector pMSCVneo containing theinsert. All gene constructs were checked by DNA sequencing.

Cell Culture and Transfection The amphotropic packaging cell lineGP+envAM12 (Genetix Pharmaceuticals, Cambridge, Mass.) and allretroviral producer lines were maintained in Dulbecco's modified Eagle'smedium (Gibco-BRL, Gaithersburg, Md.) supplemented with glutamine,penicillin, streptomycin (Gibco-BRL), and 10% fetal bovine serum(Gibco-BRL). Using Effectene Reagent (Qiagen, Valencia, Calif.), vectorDNA was transfected into GP+envAM12 packaging cells and selected withG418 (400 ug/ml; Gibco-BRL).

Enrichment and Cloning of Packing Lines by Affinity Column Theretroviral producer lines were affinity enriched using MACS goatanti-rat IgG MicroBeads on the MiniMACS system (Miltenyi, Auburn,Calif.). In brief, the transduced packing lines were reacted with 2E9(10 ug per 10⁶ packing cells) on ice for 30 minutes, washed, applied tothe anti-rat column, and eluted according to manufacturer's protocol.Cloning was done by limiting dilution. scFv expression on producerclones were monitored by flow cytometry (FACSCalibur, Becton DickinsonImmunocytometry Systems, San Jose, Calif.) using anti-idiotypicantibodies 2E9 or 1E12. Virus-containing supernatant was used to infectK562 cells, and gene transduction was measured by scFv surfaceexpression.

Enrichment and Cloning of Packing Lines by FACS sorting Cell sorting wascarried out using a Cytomation MoFlo digital cell sorter (CytomationInc., Fort Collins, Colo.), selecting for the brightest (0.1%)2E9-reactive cells, and seeded into 96-well plates at 10 cells per well.

Peripheral Blood Mononuclear Cells (PBMC) Peripheral blood from normalvolunteers and patients were obtained aseptically with informed consentaccording to the guidelines of the Institutional Review Board ofMemorial Sloan-Kettering Cancer Center. PBMC were isolated bycentrifugation on Ficoll (density, 1.077 g/ml) for 30 min at 25° C. andwashed twice with PBS. PBMC (10⁶/ml) were cultured in RPMI 1640supplemented with 10% human AB serum (Gemini Bio-Products, Woodland,Calif.), 50 μM 2-mercaptoethanol, 2 μM L-glutamine, and 1%penicillin-streptomycin (Gibco-BRL), and activated with solid phaseanti-CD3 (1 μg/ml; clone OKT3; PharMingen, San Diego, Calif.) andanti-CD28 (1 ug/ml; clone CD28.2; PharMingen) MoAbs for 3 days at 37° C.before retroviral transfection.(Koehne, 2000 #5893)

Retroviral Transduction Protocol PBMC or K562 were suspended at 1-5×10⁵cells/ml of freshly harvested supernatant from retroviral producercells, containing 8-10 ug/ml hexadimethrine bromide (polybrene,Sigma-Aldrich), centrifuged at 1000×g at room temperature for 60minutes, before culturing in 12-well tissue plates overnight. The viralsupernatant was then aspirated and fresh IMDM (Gibco-BRL) mediumcontaining 100 U/ml of IL2 and changed approximately every 5 days tomaintain a cell count between 1-2×10⁶ cells/ml. (Koehne, 2000 #5893)Transfected cells were cultured in wells coated with anti-idiotypicantibody 2E9, for 2 consecutive days each from weeks 3 to 7, and thentransferred to plates freshly coated with 2E9 at every 3 weeksintervals.

Real-Time Quantitative PCR Real-time quantitative PCR for scFv gene copynumber and RT-PCR for mRNA were performed on cryopreserved lymphocytesamples using ABI Prism 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) as previously described. (Mora, 2001#6436; Cheung, 2001 #5733) β-actin was the endogenous control for DNA,whereas glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for mRNA.Primers and probe for scFv were designed using the applications-basedprimer design software Primer Express (Applied Biosystems, ABI). Theprimers and probes for β-actin and GAPDH were from ABI.

8H9scFv sense primer: 5′-CAAATATGCTTCCCAATCCATCT-3′ (SEQ ID NO. 9)antisense primer: 5′-ACTGAGAGTGAAATCTGACCCTGAT3-′ (SEQ ID NO. 10) Probe:FAM-5′-TCCCCTCCAGGTTCAGTGGCAGTG-3′-TAMRA (SEQ ID NO. 11) β-actin senseprimer: 5′-TCACCCACACTGTGCCCATCTACGA-3′ (SEQ ID NO. 12) antisenseprimer: 5′-CAGCGGACCCGCTCATTGCCAATGG-3′ (SEQ ID NO. 13) Probe:FAM-5′-ATGCCC-TAMRA-CCCCCATGCCATCCTGCGTp-3′ (SEQ ID NO. 14) GAPDH senseprimer: 5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO. 15) antisense primer:5′-GAAGATGGTGATGGGATTTC-3′ (SEQ ID NO. 16) Probe:VIC-5′-CAAGCTTCCCGTTCTCAGCC-3′-TAMRA (SEQ ID NO. 17)

DNA and mRNA were extracted from cryopreserved lymphocyte samples andprocessed as previously described. (Mora, 2001 #6436; Cheung, 2001#5733) Every PCR run was in duplicates and included a 5-point standardto generate a standard curve for scFv and for its correspondingendogenous control, plus a no template control. scFv standard wasprepared from purified plasmid DNA, while β-actin and GAPDH standardswere purchased from ABI. For DNA samples, scFv copy number wasnormalized by the β-actin level. For cDNA samples, scFv transcript wasnormalized to that of GAPDH. The variation in the quantitation fromexperiment to experiment was within 15%. Western blotting was carriedout on lysate of scFv-modified T-cells using murine monoclonal anti-zetaantibody (BD Biosciences, Pharmingen, San Diego, Calif.; clone 8D3, 1ug/ml final dilution) and HRP conjugated goat-anti-mouse affinitypurified antibody (Jackson Immunoresearch, 1:1000 final dilution) aspreviously described. (Maher, 2002 #6433)

Cytotoxicity Assay Neuroblastoma targets NMB-7 and LAN-1, orrhabdomyosarcoma target HTB-82 were labeled with Na₂ ⁵¹CrO₄ (AmershamPharmacia) at 100 uCi/10⁶ cells at 37° C. for 1 hour. After the cellswere washed, loosely bound ⁵¹Cr was removed by washing. 5000 targetcells/well were admixed with lymphocytes to a final volume of 200μl/well. Following a 3 minute centrifugation at 200×g, the plates wereincubated at 37° C. for 4 hours. Supernatant was harvested usingharvesting frames (Skatron, Lier, Norway). The released ⁵¹Cr in thesupernatant was counted in a universal gamma-counter (PackardBioscience, Meriden, Conn.). Percentage of specific release wascalculated using the formula 100%×(experimental cpm—background cpm)/(10%SDS releasable cpm—background cpm), where cpm are counts per minute of⁵¹Cr released. Total release was assessed by lysis with 10% SDS(Sigma-Aldrich), and background release was measured in the absence ofcells. The background was usually<30% of total for these cell lines.

ELISPOT Assays 96-well PVDF plates (MAHA S4510, Millipore, Bedford,Mass.) were coated with 100 ml of anti-IFNγ monoclonal antibody (10μg/ml, Endogen, Woburn, Mass.) overnight at 4° C.

The plates were washed with RPMI 1640 and then blocked for 1.5 hour at37° C. with IMDM supplemented with glutamine (Gibco-BRL), penicillin,and streptomycin (Gibco-BRL), and 10% pooled human AB serum (Gemini). Totransduced human lymphocytes (10⁵/ml in medium containing 10% humanserum, and 100 μl/well) tumor targets were added at variouseffector:target ratios and cultured for 26 hr at 37° C. in 5% CO₂.Plates were washed free of cells and reacted with biotinylated anti-IFNγ(2.0 μg/ml) for 3 hr at room temperature, before washing and reactingwith a 1:1000 dilution of streptavidin-HRP conjugate (ZymedLaboratories, South San Francisco, Calif.) diluted in PBS containing0.5% BSA for an additional 1-2 hr at room temperature. The colorimetricsubstrate was 3-amino-9-ethyl-carbazole Sigma-Aldrich) at 0.33 mg/ml in50 mM sodium acetate buffer (pH 5) containing 0.015% hydrogen peroxide.After incubation at room temperature for 8 min, the color reaction wasstopped by rinsing the plates under running tap water and Elispotscounted under a microscope.

Adoptive cell therapy of human xenograft in immune deficient mice CB-17SCID-Beige mice were purchased from Taconic (Germantown, N.Y.). Twotypes of tumor models were used, a Winn assay (Wang, 1980 #6517) and anestablished tumor model. In the Winn assay, tumor cells (10⁶ cells) weremixed with T-cells at various tumor-lymphocyte ratios and planted in 100ul of Matrigel (BD BioSciences, Bedford, Mass.) subcutaneously.Following implantation, tumor sizes (product of orthogonal diameters)were measured. In established tumor model, tumor cells (2×10⁶ cells)alone were planted subcutaneously. Here, cell therapy was started ingroups of 5 mice per cage when tumor diameter reached 0.8 cm, usually by1-2 weeks of tumor implantation. Mice received 5 weekly intravenousCIR-gene modified lymphocyte injections by retroorbital route, 2×10⁶ perinjection together with 500 U of IL-2 ip. 50 ug of anti-idiotypic orcontrol antibody was administered ip 3 days after each lymphocyteinjection. Tumor sizes were measured twice a week. Experiments werecarried out under an IACUC approved protocol and institutionalguidelines for the proper, and humane use of animals in research werefollowed.

Statistical Analysis Data were calculated as Mean +/− SEM. Differencesbetween treatment groups were tested for significance (<0.05) by studentt-test.

Results

Construction of sc8H9-CD28-hTCR-ζ-pMSCVneo Using syntheticoligodeoxynucleotide primers 355S, 355A for the hCD8a leader -scFv-CD28,365S, 365A for scFv8H9, and 379S, 379A for hTCR-ζ-chain, the genehCD8-leader-8H9scFv-hCD28Tm-hCD28cyto-TCRζ was constructed,sequence-verified and transfected into the amphotropic packaging lineGP+envAM12, and selected in G418. Enrichment and cloning of producerlines by affinity chromatography and cell sorting The retroviralproducer lines were affinity-enriched using MACS goat anti-rat IgGMicroBeads on the MiniMACS system. Following each enrichment, viralsupernatant from the producer line was used to infect the indicator cellline K562. Surface 8H9-scFv expression on both the producer lines andthe transfected K562 (4 days after infection) were measured byimmunofluorescence using anti-idiotypic antibody 2E9. With eachsuccessive affinity enrichment (FIGS. 7A and 7C) of producer line andsubsequent successive subcloning (FIGS. 7B and 7D), the surfaceexpression (mean fluorescence) of 8H9-scFv increased and became morehomogeneous for the producer clones (FIGS. 7A and 7B) as well asindicator line K562 (FIGS. 7C and 7D). Table 1 summarized the length oftime (in weeks) required to enrich for scFv-positive producer cell line.

Retroviral transduction of primary human peripheral blood mononuclearcells Following in vitro activation with anti-CD3 and anti-CD28, primaryhuman PBMC were infected with the virus from producer line supernatantby centrifugation at 1000×g for 60 minutes at room temperature. UsingPBMC from normal volunteers, the in vitro requirement of IL2 andanti-idiotypic antibody for lymphocyte expansion was studied (FIG. 8).On day 10 after gene transduction, 17-40% of cells became scFv-positiveby FACS analysis. By day 15, 75-80% became positive and by day 24, 99%of the cells became positive. This clonal evolution to homogeneity wasfound in CD4+, CD8+ and the small CD56+ populations. IL-2 concentrationof 50 to 100 U/mL appeared optimum, and anti-idiotypic MoAb 2E9 wasabsolutely necessary to maintain prolonged T-cell growth (FIG. 8). Theseexperiments were repeated twice with similar results. In the presence of100 U/ml of IL2 and solid-phase anti-idiotypic antibodies, PBMC from 4patients with stage 4 neuroblastoma off chemotherapy and 4 separatespecimens from two normal volunteers, were expanded in vitro followingCIR-gene transduction (FIG. 9). Continual expansion (103 to 108 fold)was achieved after 150-200 days of culture, with a doubling time rangingfrom 5 to 10 days. 8H9scFv average gene copy number, transcript level,and surface expression were studied in these samples (FIG. 10). ThescFv-positive population enriched quickly during the first 20 days ofculture in the absence of 2E9 (FIG. 10A). As expected, the gene copynumber and transcript level also plateaued with similar kinetics (FIG.10B). When the scFv-positive population became >95%, an average of 4.5gene copies per cell (range 2-9) was detected, which remained relativelystable throughout the extensive length of in vitro culture. ScFvexpression was typically >95% throughout 6 months of culture (FIG. 10A).By western blot analysis, the scFv-CD28-zeta chimeric protein wasprimarily a tetramer (MW˜210 kD) under nonreducing conditions and amonomer of 54 kD in the presence of 2-mercaptoethanol. The proportion ofCD8+ cells versus CD4+ cells increased steadily to >50% by day 40 ofculture, and decreased slowly over 3-4 months. At concentrations of IL-2<50 U/mL, CD4+ cells outgrew the CD8+ population even faster (data notshown). T-cells expanded in the presence of anti-CD3, anti-CD28 and IL-2(Koehne, 2002 #6442) were unable to kill HTB-82 cells in vitro (data notshown).

Transduced lymphocytes mediated non MHC-restricted antigen-specificcytotoxicity in vitro against neuroblastoma and rhabdomyosarcoma celllines In vitro cytotoxicity against NMB-7 (FIG. 11A) and LAN-1 (FIG.11B) neuroblastoma, or rhabdomyosarcoma HTB-82 (FIG. 11C) wereefficient. Antigen-dependence was demonstrated by the total inhibitionof cytotoxicties by MoAb 8H9 (FIG. 11) and anti-idiotypic antibody 2E9(data not shown). Daudi cell line (FIG. 11D) was not killed because itwas antigen-negative. This cytotoxicity was independent of target HLAexpression or HLA types (data not shown). Unmodified lymphocytes fromthe same donor, cultured under the same conditions (100 U/ml of IL2),did not show antigen-specific killing (FIG. 11). Control (5F11scFv) CIRmodified lymphocytes also did not show antigen-specific killing of HTB82(data not shown). In Elispot assays, IFN-γ secretion was detected whentransduced lymphocytes were stimulated with antigen-positive tumors(NMB7 and HTB82) but not antigen-negative controls (Daudi, data notshown).

Adoptive cell therapy of rhabdomyosarcoma xenograft in SCID mice. Humanrhabdomyosarcoma is strongly reactive with 8H9, but not with 5F11(anti-GD₂) antibodies. 5F11scFv-CIR contained the same CD28-TCRζconstruct used for 8H9scFv-CIR. 8H9scFv-CIR gene-modified lymphocytessuppressed HTB82 tumor growth, when mixed at 1:0.5 (tumor to T-cell),1:1 or 1:10 ratios at the time of tumor implantation (FIG. 12). Whileall the mice in control group (tumor alone) or irrelevant T-cell(5F11scFv-CIR gene-modified lymphocytes) group developed rapid tumorgrowth, in the presence of specific T-cells (8H9), tumor was completelysuppressed. When anti-idiotype 2E9 was injected q 3 days×3 after tumorimplantation, the anti-tumor effect was substantially reduced, incontrast to control antibody 1G8 or saline control. This inhibitoryeffect of 2E9 on the effector phase was consistent with in vitrofindings (FIG. 11). However, when 8H9scFv-CIR gene-modified lymphocyteswas tested in an established tumor model, the growth sustaining functionof specific anti-idiotypic antibody became more apparent. Hereexperiments were initiated when tumors grew to around 0.8 cm diameter.Control groups were injected with either (1) no cells plus 2E9 ip, (2)5F11scFv-CIR modified lymphocytes intravenously plus anti-idiotype 1G8(specific for 5F11 idiotype) ip or (3) 8H9scFv-CIR modified lymphocytesintravenously plus A1G4 (irrelevant anti-idiotypic antibody) ip.Suppression of tumor growth was most significant with lymphocytestransduced with the 8H9scFv-CIR gene (o) (FIG. 8, p<0.05), and only ifthe specific anti-idiotype 2E9 was administered. 5F11scFv-CIR modifiedlymphocytes or 8H9scFv-CIR plus A1G4 did not show significant anti-tumoreffect when compared to control. This in vivo effect of gene-modifiedlymphocytes was demonstrated in 3 separate experiment.

Discussion

We have demonstrated that primary human lymphocytes could be stablytransduced with a scFv-CD28-ζ fusion gene carried by a retroviral vectorto express surface scFv. Anti-idiotypic antibody directed at the scFvfacilitated the cloning of the producer cell line and monitoring of geneexpression. These CIR-gene modified lymphocytes could proliferate in thepresence of anti-idiotypic antibody to undergo 10⁶ expansion in bothCD4+ and CD8+ populations over a period of 6 months. These cellsresponded in an antigen-specific manner in vitro by cytokine release andtumor cytotoxicity. By virtue of their near 100% CIR expression, theywere more efficient than T-cells activated in the presence ofanti-CD3/anti-CD28 and IL2. They effectively inhibited tumor growth in axenograft tumor model in a Winn assay as well as in an establishedsubcutaneous tumor model where T-cells were injected intravenously. Genetransduction was successful whether lymphocytes were derived from normalvolunteers or patients. Our data suggest that although the CIR alonepermited survival of transduced lymphocytes during the first 3 weeks,anti-idiotypic antibody was necessary for proliferation beyond thisinitial period. Several observations on the scFv-modified T-cells werenovel: the chimeric immune receptor homodimerized to a tetrameric form,T-cells expressing CIR demonstrated growth and survival advantage,anti-idiotypic antibody could inhibit effector phase during tumorkilling in vitro and in Winn assay, but enhanced tumor suppression inthe established tumor model.

The use of retroviral vectors to transduce chimeric immune receptorsinto primary human lymphocytes has been limited by the low gene transferefficiency when viral supernatant infections were carried out. Transferrates into primary human T cells using amphotropic virus ranged from 1to 12%. (Bunnell, 1995 #4550) Several strategies were explored toincrease the transduction rates to 20-50%. These include: (1) usinggibbon ape leukemia virus (GaLV strain SEATO) pseudotyped virions,(Miller, 1991 #3388; Lam, 1996 #4553; Krause, 1998 #3956) (2)coculturing producer and target cells, (Bonini, 1997 #4716) where theclinical safety was of some concern, (3) using phosphate depletionfollowed by centrifugation and incubation at 32° C., (Bunnell, 1995#4550) (4) adding fibronectin CH296 to enhance virus/lymphocyteinteractions. (Pollok, 1998 #4506) More recently, Eshhar et al describeda dicistronic construct consisting of scFv-CD28-γ and green fluorescentprotein (GFP), where the latter was used to monitor gene transductionand to enrich producer line. (Eshhar, 2001 #5665) In the inventor'sstudy, we used anti-idiotypic antibody to select for high surfacescFv-expressing producer lines with improved efficiency of genetransduction. More importantly, lymphocytes transduced by CD-28-ζchimeric fusion receptors could survive and proliferate in the presenceof the anti-idiotypic MoAb maintaining their monoclonality with respectto scFv expression, in both the CD4+ and CD8+ populations. Thesereceptors mediated antigen-specific cytokine release and cytotoxicitythat was non-MHC restricted. Whether NK cells (CD56+ population) couldacquire similar abilities will need further studies, since CD28signaling in these cells was only rarely documented. (Galea-Lauri, 1999#5609) Using this anti-idiotypic antibody strategy with minormodifications, we have successfully extended these findings to theG_(D2) antigen system (unpublished data). Recent studies havedemonstrated the potential of CIR in retargeting EBV-specific cytotoxicT lymphocytes, (Rossig, 2002 #6479) a potential new source of effectorcells that could persist and function long term after their transfer tocancer patients. We have also successfully transduced these scFv-CIRgenes into EBV-specific cytotoxic T-cell populations (Koehne, 2000#5893) to permit their in vitro clonal expansion of 10⁶-fold in 5 months(unpublished data).

The advantage of using anti-idiotypic antibody for affinity purificationand for clonal expansion of gene-modified lymphocytes are many fold.Being immunoglobulins, their structure, stability, biochemistry aregenerally known. This is in contrast to natural antigens where eachindividual system has its unique and often difficult-to-predictproperties. As surrogate antigens, anti-idiotypic MoAb are ideal forstandardization and quality control, especially for initial clinicalinvestigations of carbohydrate antigens or when the nature of theantigen is not fully understood. To prepare polyclonal CTLs specific fora tumor target, lymphocytes have to be pulsed periodically in vitro withthe tumor cells. (Koehne, 2000 #5893) The possibility of tumorcontamination raises safety and quality control issues. Moreimportantly, TCR ligation usually leads to activation-induced celldeath. (Lenardo, 1999 #6439; Beecham, 2000 #6440) In CIR technology,scFv-CD28-ζ and scFv-CD28-γ constructs recruit costimulation to sustainT-cell survival. (Alvarez-Vallina, 1996 #4698; Beecham, 2000 #6440;Maher, 2002 #6433; Eshhar, 2001 #5665; Haynes, 2002 #6477) In ourstudies, anti-idiotypic antibodies stimulated T-cell proliferation andsurvival. Another advantage of anti-idiotypic MoAb is its ability tomark the clonal population of target-specific lymphocytes. Althoughtetramers can mark TCR and T-cell clones, identity of the peptideantigen is required and tetramer technology is not widely available.Furthermore, anti-idiotypic MoAb can mark T-cell clones in vivo whenradiolabeled, an option not yet possible with tetramers. Finally, thepotential of anti-idiotypic MoAb to activate transduced lymphocytes invivo is appealing, especially when tumor cells are poorly immunogenic,or when they are scarcely distributed. The observations of theinhibitory effect of anti-idiotype in the Winn assay were consistentwith their in vitro inhibitory effects during the effector phase.However, in the established tumor model, anti-idiotype was able toenhance tumor suppression. Given its ability to sustain CIR-modifiedT-cell growth in vitro, a likely explanation was a similar supportivefunction in vivo in the established tumor model. One could speculatethat anti-idiotype might also enhance the homing properties of thesegene-modified T-cells. Clearly, a better understanding of in vivo homingproperties and proliferative capacity of transduced cells in thepresence or absence of anti-idiotype will be needed.

Previous studies suggest that the choice of the appropriate spacer(between scFv and signaling molecule), transmembrane domain and thesignaling molecules were important. (Patel, 1999 #4695) That8H9scFv-modified T-cells survive and proliferate in the presence ofspecific anti-idiotype and kill antigen-positive tumor cells arguestrongly that the CD28 trans-membrane domain in this CIR design does notrequire a CD8 hinge, permitting effective interaction with soluble aswell as cell-bound antigens. These results agreed with those recentlyreported by Maher et al. (Maher, 2002 #6433) It is of interest that inthe absence of anti-CD3/CD28 antibody activation, gene-modifiedlymphocytes had consistent survival advantage during the first 3 weeksin culture, even without anti-idiotype. Since these fusion proteins canhomodimerize, (Krause, 1998 #3956; Maher, 2002 #6433) signaling throughspontaneous oligomerization may have provided initial survival advantageon gene-modified lymphocytes, although growth could not be sustainedunless anti-idiotype is provided. Although the total increase in T cellnumber is comparable to anti-CD3/CD28 mediated in vitro expansion (Maus,2002 #6438) the rate of increase is slower (2 to 3-fold), withsignificant cell loss during the first 3 weeks. It is possible that thetransduction protocol can be improved to reduce direct toxicity from theviral supernatant. Signaling may also be improved by the addition of ahinge or the adoption of other trans-membrane domains. (Fitzer-Attas,1998 #5955; Patel, 1999 #4695; Jensen, 1998 #4699) Moreover, usingdomains or molecules (wild type or genetically modified) furtherdownstream in the T-cell activation pathway might potentially increasesignaling, or even overcome the T-cell defects commonly found in cancerpatients. (Eshhar, 1998 #5952)

The choice of tumor system and antigen target will likely determine theclinical success of CIR strategy. Primary lymphoid tumors such as B-celllymphomas have distinct attributes. T-cells have an innate tropism tolymphoid tissues. These tumors also have unique tumor antigens withhomogeneous expression that do not modulate from the cell surface (e.g.CD20). Furthermore, these B-cell tumors express costimulatory molecules.(Jensen, 1998 #4699) In contrast, most solid tumors lack theseattributes. However, metastatic cancers in lymph nodes, blood and bonemarrow are unique compartments where CIR technology may be applicable.Depending on the compartment, targeting of T-cells may require differentchemokine receptors or adhesion molecules. For example, while L-selectinis required for homing to lymphoid organs, its role for trafficking toother metastatic organs such as marrow is less well defined. The preciseevaluation of the quantity and persistence of these cells in vivo, aswell as their distribution and function within tissues is likely to becritical. (Yee, 2001 #5674; Ma, 2002 #6437) In studies of T-celltherapy, this is of particular importance since many infused cells willundergo activation-induced death in vivo, (Lenardo, 1999 #6439; Beecham,2000 #6440; Xiaoning, 1999 #5677) or immune elimination of gene-modifiedcells may occur, especially following repeated injections. (Riddell,1996 #5963) The development of sensitive, accurate and reproduciblemethods to quantify gene-marked cells in peripheral blood and tissuesare essential for defining the long-term fate of adoptively-transferredcells. While PCR and quantitative RT-PCR methods are ideal for studyingtissues extracts, anti-idiotypic MoAb will provide useful a tool toenumerate individual scFv-positive cells in blood, marrow and tumor. Inaddition, noninvasive imaging methods using radiolabeled anti-idiotypicMoAb may also be possible. Similar to the marker gene HSV-tk that allowscells to be tracked and quantified by the substrate ¹³¹I-FIAU or¹²⁴I-FIAU, (Koehne, 2000 #5631) anti-idiotypic MoAb labeled with either¹³¹I or ¹²⁴I can also take advantage of instrumentation and softwaredeveloped for SPECT and PET/micro-PET imaging, respectively. These toolscan provide unprecedented precision and dynamic information on celltraffic in patient trials.

Retroviral vector MSCV carrying the gene for either 8HscFv-CD28,8HscFv-CD28-ζ, or 5F11-scFv-CD28-ζ was transfected into packaging linesPG13 or GP+envAm12. The producer lines were then subcloned, affinitypurified or FACsorted as detailed in Materials and Methods. The producerlines were analyzed for scFv expression by flow cytometry on day 4 aftergene transduction.

Third Series of Experiments

ScFv-Modified Lymphocytes for Tumor Targeting

The plasticity of adult stem cells offers great promise in cell-basedtherapies. Hematopoietic stem cells give rise to all blood cells andhave been used to treat serious blood disorders, malignant disease, andinherited diseases. These cells can differentiate into cardiac musclecells, vascular cells, lung epithelia, neural cells, glial cells andother cell lineages. Developing tools to study both adult and embryonicstem trafficking in cellular therapies will provide a criticalunderstanding of the real potentials and limitations of theseapproaches. While technical difficulties in gene modification of humanstem cells have yet to be overcome, the human lymphocyte is a usefulmodel to explore various in vivo imaging receptors, their targetingcapacity, as well as the molecular biology and biochemistry of tracelabeling methods.

Antibody-based targeting exploits the molecular specificity of theimmune system. Utilizing single chain v-fragment (scFv) derived frommonoclonal antibodies, chimeric immune receptors (CIR) can now bepermanently transduced into primary human T-cells to redirect them tothe specific antigen. In the last grant period we developed a technologybased on anti-idiotypic antibodies to improve the rapid cloning ofefficient producer lines for gene transduction. Using anti-idiotypicantibody as antigen surrogates, the propagation and expansion of theseCIR-modified T-cells in vitro is highly reproducible. In thiscompetitive renewal, we propose to compare the three imaging genesHSV1-tk, hNIS, and somatostatin receptor type 2 (SSTR2) to study T-celltrafficking. We will take advantage of the large experience insomatostatin receptors and ligands, plus the recent development of ⁶⁸Gafor PET dosimetry studies. We will determine the biologic parametersthat determine labeling of these cells, the radiobiologicalconsequences, the minimum number of cells that can be detected at tumorsites, as well as the validation of quantitative methods of measurementmodels. We plan to test the hypothesis that substantial improvements inT-cell targeting efficiency is possible if CD4+ T-cells can bepretargeted to the tumor site, and if professional killer cells areused. The availability of a high-resolution animal scanner, the MSKCCMicroPET, plus the animal micro-CT will facilitate these studies. Wewill also benefit from prior developments under related DOE grants,which include 1) practical methods for production of ⁶⁸Ga and ¹²⁴I; 2)the quantitative PET imaging of positron-emitting radionuclides withcomplex spectra, such as ⁶⁸Ga and 124I; and 3) a method for highlyselective labeling of genetically modified tumor-specific immune cells,using the positron labeled tracer ¹²⁴I-fluoroiodo-arabinosyl-uridine(FIAU).

Objectives

Although cell-therapy using stem cells and lymphocytes have greatclinical potentials, their trafficking patterns and integration intotissues especially in real-time are not well understood. Noninvasivemethods to help fill this knowledge gap remains a critical priority.Human T-lymphocytes are potent vehicles in tumor targeting. Retroviralvectors can permanently gene-modify their cell-surface receptors totarget to specific tissues. As the sophisticated homing biology ofT-lymphocytes becomes elucidated, clinical application of adoptive celltherapy has gained wider attention. We used scFv gene-modifiedT-lymphocyte as a cell-therapy model to study the pharmacokinetics oftheir survival and proliferation ex-vivo and in vivo after reinfusion.In the last grant period, we succeeded in using anti-idiotypic strategyto optimize gene transfer, survival and proliferation of T-cells exvivo. We have shown that these cells can target to tumor sites toachieve tumor control in xenograft models. We propose to usesomatostatin receptor type 2 (SSTR2) to study cellular biodistribution,and compare with HSV1-tk and sodium iodide transporter (hNIS). We willtake advantage of recent advances in quantitative PET using ⁶⁸Ga and¹²⁴I. We propose to test the hypothesis that successful pretargeting ofT-cell subpopulation can recruit other lymphoid populations to improvehoming to the target antigen and that using preprogrammed professionalkiller cells can further improve targeting efficiency.

Specific Aim 1: Comparison of marker genes for lymphoid cells in ex vivoand in vivo labeling

-   1.1 HSV1-tk-   1.2 Somatostatin receptor subtype 2 (SSTR2)-   1.3 Sodium iodide symporter

Specific Aim 2: Pretargeting of CD4+ T-cells to improve adoptive celltherapy

Specific Aim 3: Improving tumor homing and tumor cytotoxicity by usingprofessional T-lymphocytes (CTL) and NK92 for CIR gene-modification

-   3.1 Cloned killer cell line-   3.2 Professional cytotoxic T-lymphocytes (CTL)    Importance of the Research

The study of stem cell biology in vivo can potentially broaden ourunderstanding of human cardiovascular, lung, blood, or neuraldevelopment. The homing, proliferation and differentiation of stem cellsin vivo are not fully understood and are likely to be influenced by themicroenvironment. Studies of stem cell homing to sites of tissue injuryor specific tissue or organ sites, and the mechanisms underlying thehoming process will provide important information if stem cell therapyis to be successfully exploited for human diseases. Stem cell homingresearch can benefit from tools optimized for studying T-cell targetingto human tumors. We chose scFv-chimeric immune receptor directed atsolid tumors to explore T-cell trafficking behavior, and proposenoninvasive methods to track them in vivo.

Antigen-specific T-cells have been successfully used in adoptivetherapies in patients for viral infections and cancer. These pioneeringworks have refined the practical issues of T-cell isolation, cloning,expansion and reinfusion. Adoptively transferred donor-derivedEpstein-Bar virus (EBV) specific CTL can effectively eliminate B-cellproliferative disorders in the post-transplant period, a dramatic proofof principle for both efficacy and safety. After a 2-3 log expansionwithin the first month of infusion, these CTLs can be shown to survivefor months, a property probably important for their in vivo efficacy.Successes in this EBV-lymphoma model is due to: (1) high CTL clonalfrequency, characteristic of pathogen-based memory, (2) exquisitespecificity to a viral antigen, (3) high levels of MHC and costimulatorexpression in lymphomas, and (4) innate ability of T-cells to home tolymphoid organs. Using retroviral vector gene transfers, it is nowpossible to modify durably the genetic makeup of T-lymphocytes.Targeting them to tumors is an enticing strategy since they canproliferate and expand clonally, potentially amplifying the anti-tumorresponse, as well the tumor to nontumor ratio of the delivered entity(either cells, cell-associated protein, secreted protein or viruses).

Using anti-idiotypic reagents, scFv chimeric immune receptors (CIR)consisting of scFv-CD28-ζ-chain have been transduced into primary humanT-cells to produce readily expandable, long-lived and efficient clonalkiller cell populations. Such CIRs genes joining tumor-selective ScFv toT-cell signal transduction molecules bypass MHC requirement whilecoupling antigen-specific tumor recognition with T-cellactivation/survival. In this proposal we hypothesize that tumortargeting can be substantially improved if (1) CD4+ T cells can besuccessfully targeted first to recruit inflammatory populationsincluding CD8+ T-lymphocytes and natural killer cells, and (2) theanti-tumor effect can be increased by employing preprogrammedprofessional killer T-cells. We propose to study the trafficking ofwhole or separated T-cell subpopulations in vivo after gene marking withhuman somatostatin receptor type 2 (SSTR2), human sodium iodidetransporter (hNIS) or HSV1-tk. These lymphocytes can be imaged with[^(66/67/68)GA]- DOTA-DPhe¹-Tyr³-octreotide (DOTATOC), free ¹³¹I and¹³¹I-labeled 2′-fluoro-2′-deoxy-1-b-D-arabinofuransyl-5-iodo-uracil(¹³¹I-FIAU), respectively. Lymphocyte biodistribution and clonalexpansion in vivo will be measured by positron emission tomography (PET)using [^(66/68)Ga]- DOTATOC, 124I and ¹²⁴I-FIAU for the respectiveimaging genes. Using professional cytotoxic T-cells and cloned killerline NK92 instead of naïve T-cells for CIR gene transduction, weenvision a substantial improvement in the efficiency of gene-modifiedT-cells by virtue of their preprogramming for tumor cytotoxicity, sincethey have been selected for their repertoire of lytic enzymes, deathinducing peptides and adhesion molecules.

Background and Significance

Despite dose-intensive use of chemotherapy and radiotherapy, metastaticsolid tumors have a dismal prognosis with cure rates of <20%.¹⁻³ Ourinability to deliver specific therapy to minimal residual disease (MRD)compromises patient's chance of long-term cure.

Single chain Fv. The ability to condense the binding site by geneticfusions of variable region immunoglobulin genes to form scFv has greatlyexpanded the potential and development of antibody-based targetedtherapies.⁴⁻⁷ Using phage display libraries, scFv can now be cloned fromcDNA libraries derived from rodents, immunized volunteers, orpatients.⁸⁻¹¹ Construction of the scFv is the critical first step in thesynthesis of various fusion proteins, including scFv-cytokine,¹²scFv-streptavidin,¹³ scFv-enzyme,¹⁴ scFv-toxins,¹⁵ bispecific scFv(diabodies),¹⁶ bispecific chelating scFv,17 scFv-Ig,¹² tetravalentscFv,^(16,18) and scFv-retargeted T-cells.¹⁹

Targeting lymphocytes to tumors. Using retroviral vector gene transfers,it is now possible to modify the genetic makeup of a cell permanently.Targeting lymphocytes to tumors is an attractive strategy. Lymphocytesexecute complex tasks that antibodies are unable to perform, bycommunicating with and recruiting other inflammatory/immune cells orinitiating tumor apoptosis. More importantly, they can proliferate andexpand clonally. This latter property can potentially amplify theanti-tumor response, the tumor to nontumor ratio of the delivered entity(either cells, cell-associated protein, secreted protein or viruses),for both cancer imaging as well as therapy. Antigen-specific T-cellshave been successfully used in adoptive therapies in patients for viralinfections and cancer.²⁰⁻²⁴ These pioneering work have refined thepractical issues of T-cell isolation, cloning, expansion and reinfusion.Adoptively transferred donor-derived Epstein-Bar virus (EBV) specificCTL can effectively eliminate B-cell proliferative disorders in thepost-transplant period, a dramatic proof of principle for both efficacyand relative safety.^(22,23,25) After a 2-3 log expansion within thefirst month of infusion, these CTLs can be shown to survive for up to 18months.^(22,26) This success in the EBV-lymphoma model was due to: (1)high CTL clonal frequency, characteristic of pathogen-based memory, (2)exquisite specificity to a viral antigen, (3) high levels of MHC andcostimulator expression in lymphomas, and (4) innate ability of T-cellsto home to lymphoid organs.

Tools for tracking T-lymphocyte homing and their clonal expansion arelimited. Previous models of lymphocyte homing have utilized lymphokineactivated killer lymphocytes (LAK) or tumor infiltrating lymphocytes(TIL). In animal models, they generally showed tumor-specificlocalization. Although short-term labeling with chromium (⁵¹Cr) isroutine for isotope release cytotoxicity assays, it failed when appliedto WBC trafficking studies. Attempts to incorporate radiolabeledmetabolites and metabolite analogs (including 2-fluroro-deoxyglucose(FDG), amino acids, and nucleotides,^(27,28) to radioiodinate cellmembrane lipids/proteins, to induce phagocytosis of ^(99m)Tc- or¹¹¹In-labeled colloids,^(29,30) to trap intracellular radioactivedivalent cations (e.g. ⁵⁵Co and ⁵⁷Co)³¹ or to tag with radiolabeledMoAb,³²⁻³⁴ have met with limited success. Although ¹¹¹In-labeled WBCsare routinely used to detect sites of infection and inflammation,³⁵ andin research studies of white cell homing properties,³⁶ high specificactivity can interfere with lymphocyte functions possibly accounting forthe low % ID/gm in recent studies of tumor-sensitized lymphocytes,³⁷ orTIL³⁸ cells. More importantly, ¹¹¹In labeling is currently only possibleex vivo. No imaging agent is available to study T-cell kinetics andbiodistribution over an extended period of time.

There were several limitations in these early studies of lymphocyteimaging. Only a small proportion of cells are actually labeled. In thecase of ^(99m)Tc, its relatively short physical half-life (6 hr) limitsimaging to less than 1 day post-injection. More seriously, theselabeling methods are antigen non-specific; i.e. all cells exposed to thelabeling agent are labeled regardless of their ability to bind to thetumor target. That may partly explain the suboptimal targeting of <0.02%injected cell dose per gram. The CTL precursor frequency against humantumors (e.g. melanoma) in peripheral blood mononuclear cells (PBMC) evenafter in vitro stimulation with IL-2/IL4 is generally low (0.1% to0.003%).³⁹ In EBV-lymphoma, unstimulated peripheral blood CTL precursorfrequency is less than 0.05% and is ineffective until in vitroEBV-restimulation to 0.8-4%.⁴⁰ The low CTL precursor frequency mayaccount for many of the past failures in the studies of T-lymphocytehoming to tumors. Increasing the level of radiolabeling has limitedsuccess since more than 20 uCi ¹¹¹In/10⁸ cells is known to damage whitecell functions.⁴¹

T-bodies can redirect lymphocyte against human tumors. Adoptive celltherapy using ex vivo expanded tumor-selective T-cells can effectdramatic remissions of virally induced malignancies, a processcritically dependent on clonal frequency, where rapid exponentialexpansion of specific CTL is required.^(23,25) T-cells proliferate whenactivated (e.g. anti-CD3). However, apoptosis occurs unless acostimulatory signal (e.g. anti-CD28) is present.⁴² However, human tumortargets often lack costimulatory molecules (e.g. CD80), or overstimulateinhibitory receptors (e.g. CTL4) such that the CD28 pathway is derailed.In addition, many tumors downregulate major histocompatibility complex(MHC) molecules to escape engagement by the T-cell receptor (TCR).Through genetic engineering, chimeric immune receptors (CIR) linkingtumor-selective scFv to T-cell signal transduction molecules (e.g. TCR-ζchain and CD28) will activate lymphocytes following tumor recognition,triggering the production of cytokines and tumor lysis.^(19,43-49)T-cell can also be genetically engineered to secrete cytotoxiccytokines,⁵⁰ toxins,⁵¹ or to metabolize prodrugs.^(52,53) Geneticallyengineered T cells for adoptive immunotherapy of cancer is gaining widerattention. To date, clinical experience with gene-modified T cells hasbeen limited, and most studies are unpublished (Table 1).⁴⁹ Althoughpreclinical models generally utilized CD8+ CTLs, most clinical trialsare utilizing unseparated T cells, preselected with co-expressed drugmarker, or administered in bulk without selection to avoid targeting ofmicrobial drug resistance genes. Most of these infusions have beenrelatively well-tolerated.

TABLE 1 Clinical trials using T-cells gene modified with CIR⁴⁹ DatePhase Disease Antigen Structure Location Investigator 1995 I HIV gp120CD4-ζ NIH Walker 1996 I Ovarian CA FBP sFv-γ NCI Hwu 1997- II HIV gp120CD4-ζ Multi Hege 1998 1997 I AdenoCA TAG72 sFv-ζ Stanford Hege 1998 IAdenoCA CEA sFv-ζ Harvard Junghans 2000 I Lymphoma CD19 sFv-ζ City ofHope Jensen 2001 I Neuroblastoma L1 sFv-ζ City of Hope Jensen 2002 IRenalcell CAG250 sFv-ζ den Hoed CC Bolhuis 2002 I Melanoma GD3 sFv-ζHarvard Junghans

However, significant technologic gaps remain: (1) Gene transduction intoprimary human lymphocytes is inefficient, (2) Antigen specific T-cellscannot be easily enriched and expanded, (3) Optimal T-cell activationmay require multiple signals, and (4) Demonstration of anti-tumor effectof these human T-cells in established tumor models has been difficultand so far unsuccessful in patients.⁴⁹ T-cell activation requires twosimultaneous signals,⁵⁴ one signal provided through the TCR⁵⁵ and asecond one is a costimulatory signal.⁵⁶ T-cells get their second signalfrom their CD28 molecules which recognize B7 on APCs and tumor cells,stimulating IL-2 production; otherwise apoptosis or anergy will occur inresponse to the TCR signal alone.⁵⁶ Primary T-cells transduced with theanti-GD2 scFv-ζ-chain or scFv-γ-chain CIRs were able to killantigen-positive tumors selectively.⁴⁸ However, cell cultures could notbe maintained for longer than 8 weeks even upon stimulation withantigen-positve tumor cells. In addition, T-cell function when measuredby interferon-γ release decreased substantially during in vitro cultureto 25% over 2 weeks. The inability of Fv-ζ receptors alone to activateresting T cells was demonstrated in a transgenic mouse model.⁵⁷ On theother hand, when an anti-tumor scFv-CD28 CIR was used,⁵⁸ a functionalco-stimulatory signal was achieved. CIR with multidomains was recentlydescribed, where the intracellular domain of CD28 was ligated to the 5′end of TCR-ζ chain and introduced into Jurkat cells and primary humanlymphocytes, with the expected “two signals” when scFv was triggered bytumor cells.^(59,60) IL-2 production was 20 times more than CIR withζ-chain only. Primary mouse CD8+ T lymphocytes expressing the scFv-CD28-receptor secreted Tc1 cytokines, induced T-cell proliferation, andinhibited established tumor growth and metastasis in vivo, a processshown to be critically dependent on IFN-γ secretion.⁶¹ Not all T-cellmediated immune responses are CD28-dependent, and in humans about 50% ofCD8+ T cells are CD28-negative.^(56,62) During CD28 costimulation, whileCD4+ cells responded with sustained proliferation, CD8+ T-cells grew fora limited period only accompanied by an increase in apoptosis.⁶³ Othercostimulatory molecules include members of the TNFR superfamily,^(64,65)CD30⁶⁶ and OX40⁶⁷ for Th2, as well as CD27⁶⁸ and 4-1BB for Th1.⁶⁹ It ispossible that while chimeric receptors containing CD28 will enhance CD4+T-cell proliferation, those incorporating costimulatory molecules suchas 4-1BB could enhance CD8+ T cell and other subpopulations to expand invitro and possibly in vivo.

Progress Report

CIR gene modified professional killer cells: CTL and NK92 NK92, CD56+cell line established from the peripheral blood of a 50-year-old malewith rapidly-progressing non-Hodgkin's lymphoma (large granularlymphocytic) whose marrow was diffusely infiltrated with large granularlymphocytes (LGL),⁸⁷ kills a broad spectrum of leukemia-lymphoma andvirally infected cell lines in vitro.⁸⁸ Its remarkable tumorcytotoxicity is probably due to its unique repertoire of activating NKreceptors (NKp30, NKp46, 2B4, NKGD, E, CD28) with few inhibitoryreceptors (NKGA/B, low levels of KIR2DL4, ILT-2) commonly expressed onnormal NK cells (Table 4)⁸⁹ In addition, NK92 expresses high levels ofmolecules involved in the perforin-granzyme cytolytic pathway as well asadditional cytotoxic effector molecules including tumor necrosis factor(TNF)-superfamily members FasL, TRAIL, TWEAK, TNF-alpha, indicating theability to kill via alternative mechanisms. NK92 cells can be expandedin vitro with IL-2 with a doubling time of 24 to 36 h. IL2 was alsosuccessfully transduced into NK92 which then proliferate independentlyof IL-2 for >5 months, with concurrent increase in both in vitro and invivo cytotoxicity.⁹⁰ NK92 has been used for ex vivo purging of malignantBCR-ABL-positive CD34+ progenitor cells from stem cell autografts of CML patients.⁹¹ In phase I clinical trials, children and adults with latestage malignancies have received repeated irradiated NK92 transfusionsup to 9×10⁹ cell dose without adverse reactions.⁸⁸ Patients had noevidence of anti-NK92 immune response. However, NK92′s lytic activityagainst solid tumor targets is less predictable. 8H9-scFv-CD28-ζ hasalso been transduced into NK92 cells, and the high expressors sorted andcloned using anti-idiotype strategy as described for primaryT-lymphocytes. These gene-modified NK92 cells can efficiently kill anexpanded spectrum of tumor lines in vitro as well as suppressing humantumor xenografts in vivo.

Gene expression profile of CIR-modified T-cells and NK92 An analysis ofinflammatory chemokines, cytokines and receptors, as well asinterleukins and receptors gene expression among CIR-modified NK92 aswell as CIR-modified T-cells (cultured for 70 days, 99.7% scFv-positive,50% CD4+ and 50% CD8+, harvested during their exponential growth) wasundertaken. The GEArray Q series cDNA expression arrays (SuperArray,Bethesda, Md.) were used for these assays. In brief, cDNA probes using 5ug each of T cell RNA from CIR-gene modified blood lymphocytes, versusfresh and cultured control lymphocytes, were synthesized withbiotin-16-UTP. Hybridization at 60° C. was carried out in ahybridization chamber with constant rotation overnight. After severalwashes, chemiluminescent detection was performed at room temperature. A1:10,000 dilution of alkaline phosphatase-conjugated streptavidin wasplaced on the membrane after a 40 minute blocking step. After severalwashes, CDP-Star chemiluminescent substrate was added. The membrane wasplaced between two transparencies, and developed on X-ray film for 10seconds. Data analysis of the image was based on SuperArray software(Eisen Lab, LBNL, UCB, Calif.) and GEArray Analyzer (SuperArray). Fourgene chips were used: human inflammatory cytokine and receptor, humaninterleukin and receptor, human extracellular matrix and adhesionmolecules, human cytokines and receptors. Experiments were repeated atleast once. Gene expression values were normalized to that of GAPDH andvalues from multiple chips were averaged. CIR modified T-lymphocytesdisplayed remarkably similar profiles of interleukin plus receptor(Tables 5 and 6, minus=negative, W=weak, Y=strong expression) andchemokine plus receptor (Tables 7 and 8) as compared to culturedT-lymphocytes without CIR gene modification, consistent with theexpectation that CIR gene transduction did not substantially change thephenotypes necessary for their immune functions. Furthermore, bothgene-modified T and NK92 cells expressed common chemokines includingRANTES, and a broad spectrum of interleukins (e.g. IL4, table 4) andinterleukin receptors (e.g. IL15R, table 6) with potential importance inamplifying the anti-tumor response. In contrast to low/absent CCR7(thereby allowing T-cell to recirculate instead of docking in lymphnodes), CCR5 highly expressed for both 8H9s-scFv-CD28-ζ modified T andNK92 cells (Table 8). Since IL7 receptor was not detected among eitherT-cells or NK92, while IL15 receptor was expressed by both, IL15 may beuseful for enhancing the survival of CD8+T-cells both in vitro and invivo. Also of note was the low level or absence of IL-2 and IFN-γ whenthe cells were harvested while off anti-idiotypic antibody.

Transduction of HSV1-tk into primary human T-cells HSV1-tk is atherapeutic gene, a marker gene, as well as a suicide gene. In order toexamine the migration of genetically altered antigen-specific Tlymphocytes to tumors after adoptive transfer in vivo, we exploited thecapacity of transduced T cells expressing HSV-TK to selectivelyphosphorylate and trap in cells and incorporate into DNA radiolabeledthymidine analog 2′-fluoro-2′deoxy-1-D-arabinofuransyl-5-iodo-uracil(FIAU) (FIG. 1). Gamma camera images and autoradiographs showedselective tumor localization of ¹³¹I-FIAU-labeled HSV1-tk-transducedEBV-specific, HLA-matched allogeneic donor T cells in preclinicalmodels, achieving 1-2% injected per gram of tumor, and tumor-to-normaltissue activity ratio >100:1. In contrast to conventional cell labelingmethods which are non-selective; FIAU labeled only those lymphocyteswith the HSV1-tk transgene, yielding a highly purified and highlytarget-specific lymphocyte population. In addition, HSV1-tk transducedprimary human PBLs were sensitive to ganciclovir (0.01-0.1 uM) in vitroand in preclinical models (20 mg/kg bid×7 days) The ability ofEBV-specific HSV1-tk transduced T-cells to home and kill subcutaneousEBV lymphoma xenografts was completely removed by ganciclovir treatment,thus allowing these gene-modified T-cells to be safely removed whennecessary.

The cell-level dosimetry of lymphocytes labeled by incubation ex vivowith radioiodinated FIAU was critical since [¹³¹I]-FIAU could interferewith T cell function. In this model, the FIAU uptake (i.e. labeling) ofthe lymphocytes is expressed as the accumulation ratio (AR=cpm per gramof cells/cpm per ml of medium). The absorbed dose to the lymphocytenucleus to reference (r) time T_(r), D_(n)(T_(r)), is calculated as thesum of the medium (m)-to-nucleus (n) dose, D(n←m), and thenucleus-to-nucleus dose, D(n←n). D(n←m) was equated with the meannon-penetrating (np) radiation (i.e. β) dose, D_(np), from radioiodinein the medium (assuming the presence of the widely dispersed unitdensity cells would not significantly perturb the electron flux andtherefore the dose from radioiodine otherwise uniformly distributed inthe medium) and D(n←n) was calculated assuming instantaneous cell uptakeof [³¹¹I]-FIAU and using the recently published MIRD cell S factors.⁹²

$\begin{matrix}{{D_{n}\left( T_{r} \right)} = {{D\left( n\leftarrow m \right)} + {D\left( n\leftarrow n \right)}}} \\{\approx {D_{np} + {D\left( n\leftarrow n \right)}}} \\{= {{\Delta_{np} \cdot T^{*} \cdot {\lbrack A\rbrack_{m}/\rho}} + {{S\left( n\leftarrow n \right)} \cdot \lbrack A\rbrack_{m} \cdot M_{c} \cdot {\sum\limits_{i = 1}^{r}{{AR}_{i} \cdot {\int_{T_{1}}^{T_{1} + 1}{{\mathbb{e}}^{{- \lambda}\; p\; t}/\rho}}}}}}}\end{matrix}$where

-   -   Δ_(np)=0.405 gm-rad/μCi-hr for ¹³¹I,    -   T*=time of incubation of the cells with ¹³¹I-FIAU        -   ≈2 hr,    -   T₁=time from the start of the incubation in the        [¹³¹I]-FIAU-containing medium,    -   T_(r)=reference time from the start of the incubation in the        [¹³¹]-FIAU-containing medium for which the dose is calculated        -   ≡60 hr (typical in vivo imaging time post-injection),    -   [A]_(m)=activity concentration in medium (μCi/ml),    -   ρ=mass density        -   =1 gm/ml for both medium and cells,    -   S(n←n)=the nucleus-to-nucleus S factor (i.e. dose per unit        cumulated activity        -   =1.43×10⁻⁷ rad/μCi-hr for ¹³¹I    -   AR₁=accumulation ratio at time T_(i) from the start of the        incubation in the [¹³¹I]-FIAU-containing medium    -   M_(c)=mass of cell        -   ≡1×10⁻⁹ gm/cell as measured for T lymphocytes    -   λ_(p)=the physical decay constant of radioiodine        -   =0.0036 hr⁻¹ for ¹³¹I.

Based on the forgoing dosimetry model and as presented graphically inthe FIG. 15, the lymphocyte nucleus absorbed dose was calculated as afunction of activity concentration in the medium and the accumulationratio. To study the effect on T-cell function, [131I]-labeled FIAU wasincubated with HSV1-tk transduced T cells at 11 Ci/ml at 37° C. for 40to 120 min in increasing activity concentrations of [131]-FIAU from 1.1to 56 μCi/ml, washed and transferred to fresh ([131I]-FIAU-free) mediumfor 72 hr. and then used in a 51Cr-release immune cytotoxicity assay(low effector:target cell ratio=5). There was no demonstrable diminutionin immune function up to an absorbed dose (at the reference time of 60hr) of 1,200 cGy. At greater doses (>1,900 cGy), there was adose-dependent decrease in immune function.

Radioactive gallium labeled somatostatin analogueDOTA-DPhe1-Tyr3-octreotide (DOTATOC table 9) for positron emissiontomography imaging93 Radionuclide labeled somatostatin analoguesselectively target somatostatin receptor (SSTR)-expressing tumors as abasis for diagnosis and treatment of these tumors. Recently, aDOTA-functionalized somatostatin analogue, DOTATOC has been developed.This compound has been shown to be superior to the other somatostatinanalogues as indicated by its uniquely high tumor-to-nontumor tissueratio. DOTATOC can be labeled with a variety of radiometals includinggallium radioisotopes. Gallium-66 is a positron emitting radionuclide(T1/2=9.5 hr; βy+=56%) that can be produced in carrier free form by alow-beam energy cyclotron. SSTR targeting characteristics of66Ga-DOTATOC were studied in nude mice implanted with AR42J rat pancreastumor, and compared with 67Ga- and 68Ga- labeled DOTATOC. The labelingprocedure gave labeling yield ranged from 85-95% and radiochemical andchemical purity was >95%. In-vitro competitive binding curves and invivo competitive displacement studies with an excess of unlabeledpeptide indicates that there is specific binding of the radioligand toSSTR. Animal biodistribution data and serial microPETTM imagesdemonstrated rapid tumor uptake and rapid clearance from the blood andall tissues except kidney. Maximum % ID/g values for tumor were10.0+0.7, 13.2+2.1 and 9.8+1.5 for 66Ga-, 67Ga-, and 68Ga-DOTATOC,respectively. Calculated tumor, kidney and bone marrow doses for66Ga-DOTATOC based on biodistribution data were 178, 109 and 1.2cGy/MBq, respectively. 66Ga labeled DOTATOC can be used for PETdiagnosis and quantitative imaging-based dosimetry of SSTR positivetumors. 66Ga-DOTATOC may also be used in higher doses for ablation ofthese tumors. However, kidney is the critical organ for toxicity(tumor/kidney ratio=1.64).93

Background and Statement of Work

Specific Aim 1: Comparison of Marker Genes for Lymphoid Cells in Ex Vivoand In Vivo Labeling: HSV1-tk, Somatostatin Receptor Subtype 2 (SSTR2)and hNIS

1.1 Herpes Simplex virus I thymidine kinase (HSV1-tk), In vivo methodsfor monitoring gene-modified cells have exploited the sensitivity ofgamma-camera (SPECT) or PET imaging to detect intravenous radiolabeledcompounds that localize to the products of transferred genes. Thesegenes include enzymes that metabolize drugs (Herpes Simplex Virus-1thymidine-kinase [HSV1-tk]), transport drugs across cell membranes(sodium-iodide symporter [NIS]), and ligand-binding surface receptors(type 2 somatostatin receptor [SSTR2]⁹⁴⁻⁹⁶ and the type 2 dopaminereceptor).^(97,98) HSV1-tk gene transfer can be detected by both gammaand PET imaging using radiolabeled prodrugs (e.g. ¹²⁴I-FIAU) that becomeentrapped in the cell after phosphorylation by the kinase.⁹⁹⁻¹⁰³ Giventhe limitations of in vitro radiochemical cell labeling, a marker genethat does not interfere with T-cell function is critical forbiodistribution studies of adoptively transferred T-cells. Each of thesethree marker genes have their merits and disadvantages.

HSV1-gk SSTR2 NIS Human origin N Y Y Substrate + +++ ++++ availabilitySubstrate ± safe safe safety record PET capability Y Y Y Suicidefunction Y N N Cellular retention Y Y N Distribution cytoplasmicmembrane membrane Tissue specificity Y tumors; thyroid/ some stomach/normal salivary tissues gland

Key advantages of HSV1-tk include its suicide function and itsspecificity (i.e. not found in human solid tumors). However, there areseveral limitations: (1) To label HSV1-tk-gene modified lymphocytes invivo may need high concentrations of FIAU is needed. The safety ofiodine-labeled FIAU especially at high doses is unknown, while unlabeledFIAU itself has been linked to severe hepatic toxicity in clinicaltrials. (2) Iodine-labeled FIAU requires special and expensiveradiochemistry, (3) Nuclear location of metabolized radiolabeled FIAUcan damage cellular DNA, limiting the absolute amount of radioiodine perlymphocyte. (4) HSV1-tk is a foreign protein, potentially antigenic andallergenic. (5) HSV1-tk is an intracellular protein, the expression ofwhich is 15 hard to quantitate in live cells. (6) Suicide withganciclovir requires cell division and can be compromised because ofHSV1-tk gene deletions.¹⁰⁴

1.2 Human type 2 somatostatin receptor (SSTR2) SSTR2⁹⁴⁻⁹⁶ is a membranereceptor that can be imaged with radiolabeled peptide ligands including^(99m)Tc-P829 [NeoTect, Amersham Health, Princeton, N.J., FDAapproved],¹⁰⁵ ¹⁸⁸Rh-P829, ^(99m)Tc-P2045, and ¹¹¹In-octreotide[Mallinckrodt] which is FDA-approved for total body imaging. We choseSSTR2 because it has been used extensively in clinical imaging withreadily available radiolabeled ligands. In addition, a number ofoptimization strategy has already been designed, including intravenousL-lysine to reduce renal uptake. There are 6 SSTRs: types 1, 2A and 2B,3, 4, and 5, all belonging to the 7-transmembrane domain family ofreceptors associated with G-proteins. Human type 2 has high affinity foroctreotide, types 1 and 4 have low affinity,^(106, 107) and types 3 and5 have intermediate affinity.¹⁰⁸⁻¹¹⁰ Types 2A and 2B are alternatesplice variants where type 2A has a longer intra-cytoplasmic carboxyterminus than type 2B. SSTR2 expression has been reported in humanlymphoid and leukemia cell lines, human peripheral blood lymphocytesespecially when activated with PHA.¹¹¹ SSTR2 is the dominant receptorsubtype expressed by inflammatory cells including T-cells.¹¹²Somatostatin and its analogs specific for SSTR2 enhance adhesion ofT-cells to fibronectn.¹¹³ ¹¹¹In-pentetreotide (octreotide) was used forpredicting impending cardiac allograft rejection before endomyocardialbiopsy becomes positive.¹¹⁴ The inhibitory effect of somatostatin onlymphocyte proliferation¹¹⁵ is mediated by SSTR-5.¹¹⁶ When a panel ofoctreotide ligands were screened for their binding affinity andspecificity (Table 9), Gallium labeled-DOTATOC was chosen for ourstudies because of its high affinity and specificity towards SSTR2preferentially over SSTR5 which can interfere with the proliferation ofgene-modified T-lymphocytes.

1.3 Na⁺/I⁻ Symporter (NIS) Both rat and human NIS, a membrane-boundglycoprotein which is responsible for the thyroid gland's ability toconcentrate iodide up to 40-fold with respect to plasma, was recentlycloned,^(117,118) and its genomic structure analyzed.¹¹⁹ hNIS has 643amino acid and a proposed secondary structure containing 13transmembrane helices. NIS was upregulated with trans-retinoic acid inbreast cancer cell line MCF7.¹²⁰ Prostate cell lines transfected withhNIS linked to a PSA promoter became sensitive to radioiodinetherapy.^(121,122) Adenovirus-mediated¹²³ or retrovirus-mediated¹²⁴transfer of rat NIS into human carcinoma lines and human glioma celllines¹²⁵ enabled rapid perchlorate-sensitive radioiodine uptake, in somecases to >200 fold. Xenografted tumors injected intratumorally with thisadenovirus became iodine-avid accumulating 11% ID/gm. Prostate cancer(LNCaP) transfected ex vivo with the hNIS retained 25-30% of the totalradioiodine with a biologic half-life of 45 h (30-60 h) and producedtumor shrinkage.¹²² The slow efflux of iodide from NIS transduced cellscan be partly explained by their lack of the efflux pumppendrin,^(126,127) found exclusively in the thyroid but not other normaltissues.

Advantages and limitations of SSTR2 and hNIS to track T-cells areseveral fold. (1) Their radioligands are commercially available andinexpensive. (2) The safety and toxicities of the ligands are wellknown. (3) The bound ligands, unlike nucleotides, do not persist in DNA.(4) If transduced into T-cells, both HNIS and SSTR2 are human-derivedand less likely to be antigenic or allergenic. (5) Clinicalpharmacokinetics of the radioligands are well characterized, (6) SSTR2and hNIS are surface proteins easily monitored with fluorescent orradiolabeled peptides or monoclonal antibodies, allowing high expressorsto be potentially enriched by affinity column or flow cytometry. (7)Since it is naturally expressed by some activated T-lymphocytes, SSTR2appears compatible with normal T-cell biology.

Neither NK92 nor CIR-gene modified T-cells expressed SSTR2 or showedspontaneous uptake of ¹¹¹In-Octreotide; thus SSTR2 gene transduction isnecessary for imaging purposes. Surface receptor SSTR2 versus enzymeHSV1-tk approach have recently been compared in vitro and in vivo.Although uptake was equally good in vitro, in vivo imaging with HSV1-tkappeared inferior to SSTR2.⁹⁶ We expect radiometal labeled peptides tobe rapidly endocytosed following binding to SSTR2, and become trappedintracellularly, unlike radioiodine which is metabolized and released.One major disadvantage of SSTR2 is its presence in a large spectrum ofneuroendocrine tumors; here T-cell trafficking and tumors may not beeasily distinguishable. Nevertheless, most sarcomas¹²⁸ and high risk (incontrast to low risk) neuroblastoma¹²⁹ have low expression of SSTR2.HNIS has a clear advantage over SSTR2 since few tumors except thyroidand possibly breast cancers express this protein. Although NIS can betransfected into human cells to express functional protein, the cellularconsequences of the ectopic ion channel or iodine accrual on the humanlymphocytes are unknown. There is also the concern on the membranetrafficking of the symporter. Although the leader sequence in thepVector would enhance membrane localization of the transgene, the rateof symporter turnover could affect the amount of radioiodine uptake. Theefflux of iodide and consequently the short cellular half life can alsobe a limitation, especially if repeated imaging studies are needed.Nevertheless, this is a surmountable issue since radioactive iodine canalways be readministered. Ironically this efflux could be an advantage,since radioactive iodide is rapidly excreted and less likely to damagelymphocyte function. It is conceivable that if retention of the iodideis needed, NK92 line can first be transfected with thyroid peroxidaseenzyme to ensure organification.¹³⁰ One unique advantage of HSV1-tk isits suicide function that kills transduced cells in the presence ofganciclovir. Nevertheless, hNIS-transduced lymphocytes can potentiallybe killed by high dose of ¹³¹I or ¹²⁴I, as demonstrated in NIS-genemodified tumor cell lines^(120,122-125) and the thyroid gland.

General Plan of Work:

Comparison of HSV1-tk, HNIS and SSTR2 in gene marking of cloned killerlymphocytes

Marker Ligand or substrate Gene Gamma PET HSV1-tk ¹³¹I-FIAU ¹²⁴I-FIAUhNIS ¹³¹I ¹²⁴I SSTR2 ¹¹¹In- DOTATOC ⁶⁸Ga-DOTATOC Or ⁶⁷Ga-DOTATOC

NK92 is a cloned killer cell line with well established characteristics(see Progress Report). EGFP (green fluorescence protein) was previouslytransduced into these cell lines and cloned, now used as our indicatorline. Gene transduction will be carried out in two separate steps. Firstwe use pDisplay vector from Invitrogen (Carlsbad, Calif.) to transduceeither the SSTR2 or hNIS into NK92 cells as previously described.⁹⁶ Alight chain 5′ IgK leader sequence for membrane localization plus ahemagglutinin (HA) tag will be inserted upstream of the SSTR2 and hNISgenes.^(131,132) A stop codon will also be introduced into the 3′ end toprevent expression in addition to the carboxy-terminal tail of SSTR2 orhNIS.¹³² Since the binding domain for somatostatin is in the carboxylend of SSTR2 between domains III and VII, amino terminal tag is notexpected to interfere with receptor internalization.¹³² Full lengthSSTR2 cDNA (type A)¹³³ was obtained from Dr. S Dorosio, U. of Iowa. Fulllength hNIS cDNA (2335 bp) was kindly provided by Dr. S. Jhiang of OhioState University, Columbus, Ohio. Using anti-HA antibody (pDisplayvector), high expressors will be selected by affinity chromatography orcell sorting, and cloned in vitro. HSV1-tk was previously successfullytransduced into NK92 using a discistronic vector, and selected with NGF(low affinity receptor) (see Progress Report). We have shown that NK92can undergo multiple gene transductions and cloned without loosing itsin vitro growth and cytotoxicity properties.

Specific Methods:

Saturation binding studies with ^(66/67/68)Ga-DOTATOC Fresh cellmembrane suspension (50 ug) on ice in 10 mM HEPES (pH 7.6, 20 μg/mLbacitracin, 5 mM MgCl₂ pH 7.6) is mixed in triplicates with increasingconcentrations of ⁶⁷Ga-DOTATOC (5 pM-5 nM) either with or without 1 μMoctreotide. The mixtures are placed on an orbital shaker for 45 minutesat room temperature before being diluted with 1 mL of ice cold salinebuffer (150 mM NaCl, 10 mM Tris pH 7.4). The suspension is then rapidly(with vacuum) filtered over glass fibre filters (Whatman GF/C, presoakedin 1% BSA) and the tubes washed twice with ice cold saline (2×4 mL). Theglass fiber filters were then removed before being counted with anautomatic NaI(Tl) counter. For each data point, triplicates wereperformed. Specific binding is defined at the total binding minus thenon-specific binding (i.e. in the presence of 1 μM octreotide). The datais then analyzed by saturation curve analysis. An analogous method waspreviously used to determine the binding affinity of DOTATOC forindividual SSTRs expressed by transfected CHO cells (Table 9).

Kinetics of [^(66/67/68)Ga]-DOTATOC uptake and cellular dosimetry ofindicator killer line NK92 SSTR2 gene modified lymphoid cells will beincubated in the presence of [^(66/67/68)Ga]-DOTATOC. Followingincubation, cells will be washed twice with ice-cold medium andradioactivity measured in a γ-counter, and normalized to cell number.The human neuroblastoma cell line SKNSH transfected with hSSTR2 (kindlyprovided by Dr. S. Dorosio of U. of Iowa, Iowa) is used as a positivecontrol. The time-dependent activity concentration in the cells will becalculated. To study cellular damage on NK92 or T-lymphocytes, cells arelabeled by incubation for 2 hr with increasing radioligandconcentrations, washed and transferred to fresh nonradioactive mediumfor 72 hr, and an aliquot tested in vitro for cytotoxicity in⁵¹Cr-release (at low E:T ratio) and IFN-(production. The rest of theNK92 are grown in fresh medium for 3 days, and their cell viability andcell number assayed. We want to confirm our previous results thatcellular viability and immune function are not affected at absorbeddoses (at 60 hr) of <=1,200 cGy. In addition to checking for immunefunctions, we will also establish a dose response curve for the level ofradioactivity uptake and inhibition of lymphocyte proliferative capacityin a standard MTT assay.

Internalization and shedding of [66/67/68Ga]-DOTATOC following SSTR2receptor binding Internalization studies will be carried out using amodification of previously published methods.¹³⁴ Following[^(66/67/68)Ga]-DOTATOC binding at 4° C. (on ice) and unbound ligandsremoved by washing, cells are incubated at 0° C. or 37° C. for varioustime periods. Free ligand in the supernatant and PBS wash are counted ina γ-counter. Remaining [^(66/67/68)Ga]-DOTATOC on the cell surface areacid stripped by incubation with a buffer containing 0.05 M glycine HCland 0.05 M acetic acid (pH 2.8-3) and 150 mmol NaCl for 5 min at 0°C.¹³⁵ The fraction of internalized ligand is calculated from theremaining radioactivity divided by the initially bound radioactivity.

-   -   Total cpm=free+acid-stripped+internalized    -   Cell bound=acid-stripped+internalized    -   % internalized=100* internalized/total cpm    -   % cell bound=100* cell bound/total cpm    -   % free=100* free/total cpm

^(66/67/68)Ga Labeling of DOTATOC ⁶⁶Ga is produced by the cyclotron onsite at Memorial Sloan-Kettering Cancer Center. ⁶⁷Ga is commerciallyavailable. Five microliters of carrier-free ⁶⁷Ga (930 mCi/mL, 0.05 MHCl) is added to 40 μL of 0.3 mM NH₄OAc (pH 7) and 4 μL of 1 mM DOTATOC.The reaction mixture is placed in a water bath at 100° C. for 15 minutesbefore a 1 μL portion is removed and diluted to 2 mL with 4 mM DTPA (pH4.0). Fifty microliters of this solution is then analyzed by HPLC usingC18 column (4 μm, 3.9×150 mm) and an eluant of 1.2 mL/min 20 mM NH₄OAc(pH 4), 0-60% acetonitrile gradient over 15 minutes. Typicallyincorporation rates are in excess of 99.5%.

⁶⁸Ga is eluted from a SnO₂ based ⁶⁸Ga/⁶⁸Ge generator in 5 ml of 1 M HCl.The concentration of HCl is increased to 5 M and the solution extractedwith 2×1.5 mL diisopropyl ether. The ether fractions are pooled andevaporated under a stream of nitrogen. The concentrated ⁶⁸Ga (3-4 mCi)is then dissolved in 50 μL of 0.3 M NH₄OAc and added to 3 μL of 1 mMDOTATOC. The mixture is heated at 100° C. before a 1 μL portion isremoved and diluted to 1 mL with 4 mM DTPA (pH 4.0). The dilutedsolution is spotted onto two 10×1 cm ITLC-SG strips and developed ineither 4 mM DTPA (pH 4.0) or 1 M NH₄OAC (pH7, 50% MeOH). In the pH4 TLCsystem the ⁶⁸Ga-DOTATOC remains at the origin with any colloidal⁶⁸Ga(OH)₃ and ⁶⁸Ga-DTPA migrates with the solvent front. In the pH7system, colloidal ⁶⁸Ga(OH)₃ remains at the origin and ⁶⁸Ga-DOTATOC and6⁸Ga-DTPA move with the solvent front. Typically incorporation rates arein excess of 99.5%.

Kinetics of radioiodide uptake in NIS transfected cells and cellulardosimetry of NK92 hNIS Gene modified NK92 and T-lymphocytes will beincubated in the presence of carrier-free Na¹²⁵I (Amersham PharmaciaBiotech) and 10 μM NaI (to give a specific activity of 20 mCi/mmol),with or without 30 μM KClO₄. Following incubation, cells are washedtwice with ice-cold medium and radioactivity measured in a γ-counter,and normalized to cell number. The rat thyroid cell line FRTL-5 (fromATCC) is used as a positive control. [¹³¹I]-labeled FIAU is incubatedwith HSV1-tk-transduced-NK92. The time-dependent activity concentrationin the cells will be expressed as the accumulation ratio (see ProgressReport). Next, the NK92 cells or lymphocytes are labeled by incubationfor 2 hr with increasing radioligand concentrations, washed andtransferred to fresh nonradioactive medium for 72 hr. and an aliquotthen used in a ⁵¹Cr-release immune cytotoxicity assay (at low E:Tratio). Another aliquot will be allowed to propagate in fresh medium for3 days, and their cell viability and cell number measured. We willconfirm our previous results that cellular viability and immune functionare not affected at absorbed dose (at 60 hr) of at least 1,200 cGy.Since iodide is not sequestered in the nucleus, we expect the maximumtolerated dose to be higher for hNIS, which should improve scintigraphicimaging in vivo. A dose response curve for cytotoxicity will beconstructed.

Iodide efflux assay The dose-dependent release of activity from NK92 orlymphocytes will also be evaluated as a function of post-labeling time.At various time after the incubation of the effector cells in ¹³¹Icontaining medium and transferring to [¹³¹I]-free medium, the activityremaining in the cells and leaking into the medium are assayed. Thecontent of ¹³¹I in the supernatant is measured by γ-counter. After thelast time point, the cells are extracted with 400 μl ethanol to countresidual radioactivity. The rat cell line FRTL-5 is used as a positivecontrol.

Western Blot analysis Postnuclear membrane fractions will be preparedand western blot analysis performed using a rabbit anti-SSTR2 antibody(BioTrend, Chemicals, Destin, Fla.), using a murine monoclonal anti-NISantibody (kindly provided by Dr. J. Struck of Brahms, Berlin, Germany),or anti-HSV1-tk antibody (Dr. Tjuvajev, MSKCC, N.Y.) and horseradishperoxidase-conjugated anti-mouse IgG (Jackson Research Laboratories),and signal visualized by chemiluminescence. Quantitative analysis isperformed using the NIH IMAGE program(http://rsb.info.nih.gov/nih-image/).

FACS analysis Cells expressing the HA-tagged SSTR2 can be monitored withanti-HA antibody (12CA5, Boerhinger-Mannheim, Indianapolis, Ind., orHB-66, ATCC, Rockville, Md.) or rabbit anti-SSTR antibody (Santa CruzBiotechnology, Santa Cruz, Calif.). Cells expressing SSTR2 are firstreacted with specific antibody or IgG control, washed and then reactedwith FITC-goat anti-rabbit (if primary antibody is rabbit) or FITC-oatanti-mouse (if primary antibody is mouse monoclonal) affinity purifiedantibody (Jackson). Propidium iodide (10 μg/ml) is used to mark damagedcells, and excluded from the analysis. SKNSH neuroblastoma cell linewill be used as the positive control for SSTR2 expression. Thefluorescence of 5000-10000 cells/tube is assayed using the FACSCaliburcytofluorometer (Becton Dickinson). Cells expressing HA tag can bemonitored with anti-HA antibody. Alternatively, hNIS without HA can bemonitored with the MoAb from Brahms, Germany.

Quantitative measurement of T-cells in tissue sections In order todetermine quantitatively the number of lymphocytes trafficking to thetumor site, we plan to perform 2 kinds of experiments: (1) extractingsingle cells from tumors and (2) by radiotracer technique. Single cellsuspensions are prepared from a known weight of tumor using collagenaseenzyme mixtures.

After ficoll-gradient to remove debris and dead cells, the number ofgene-marked lymphocytes are quantitated by flow cytometry using EGFP(NK92 only), anti-idiotypic antibody and marker-gene specificantibodies: anti-HA (for SSTR2), anti-hNIS, and anti-NGFR (HSV1-tk)antibodies. To avoid collagenase/protease modification of surfaceproteins fresh frozen tissue sections will also be analyzed by directfluorescence (EGFP, NK92), or indirect immunofluorescene using specificantibodies. The relationship between cell dose injected and the numberof T-cells/gm of tumor will be determined. Quantitative autoradiographycan also be performed although they need to be correlated withhistology. For cells carrying HSV1-tk gene, they can be labeled with¹³¹I-FIAU, or SSTR2 gene with ¹¹¹In or ⁶⁷Ga-DOTATOC, and hNIS labeledwith 125I for radiotracer experiments.

Imaging and quantitative measurement of tumor infiltrating T-cells SCIDmice xenografted with human tumors are injected i.p. with 2 ml of 0.9%NaI solution to block thyroid uptake of radioactive iodide. Gamma cameraimaging and SPECT are performed with a dual-headed ADAC Genesys gammacamera (ADAC, Milpitas, Calif.) equipped with a HEHR collimator.Sequential images are obtained at 1, 4, 18-24 h after cell injection.PET images can provide highly accurate quantitation of radiolabeled celldistribution within the body. The PET protocol consists of scanning at1, 4, 18 hr post infusion. For ex vivo labeling ⁶⁶Ga-DOTAOTC (T-1/2=9.5h) or ¹²⁴I can be used. For in vivo labeling, shorter half life isotopesuch as ⁶⁸Ga (T-1/2=68 min) will also be tested. Images will bereconstructed and attenuation corrected. Transaxial and sagittal sliceswill be studied in order to ascertain the uniformity of the radiolabeldistribution. With micro CT fusion, for each time point, the specificactivity of isotope per volume plotted over time can be calculated. Timeactivity graphs will be decay corrected for isotope in order to obtain abiological clearance curve.

Cell labeling in vivo after homing to tumor sites To test the concept ofimaging scFv-CIR modified lymphocytes, animals are treated with NaI i.p.to block the thyroid uptake. No-carrier-added ¹³¹I, ¹²⁴I (for hNIS),¹³¹I-FIAU or ¹²⁴T-FIAU (for HSV1-tk) and ¹¹¹In- DOTATOC or^(66/67/68)Ga- DOTATOC will be injected iv at 24 h, 48 h, or 72 h afterT-cell injection, depending on when the maximal homing occurs frombiodistribution experiments. Tumors in mice will be imaged by gamma(planar or SPECT) or PET where appropriate. Biodistribution studies atvarious time points will be done by tissue counting. Tissues will alsobe analyzed (direct and indirect fluorescence plus QAR.

Retroviral dicistronic construct Although imaging gene and CIR can beseparately introduced into established killer lines like NK92, forprimary human T-cells, both marker and CIR genes have to be transducedsimultaneously. We utilize the bicistronic vector that contains the CIR,internal ribosome entry-site sequence (IRES), and SSTR2 or hNIS orHSV1-tk. Both SSTR2 and hNIS genes (with their leader or tag sequencesfrom the pDisplay vector from Invitrogen, Carlsbad, Calif.) are firstPCR amplified with appropriate primers (to make Sal1- SSTR2 - Not1) toswap with HSV1-tk in pIRES. Zeta chain will be inserted into MCS ofpIRES using the fragment Xho1-zeta-Mlu1. After digestion with Xho1 andNot1, the ζ-IRES-HSV1-tk is swapped with ζ-chain in the scFv-CD28-ζconstruct using Xho1 and blunt end ligation.

Anti-idiotype enrichment of viral producer line by cell sorting Buildingon initial successes with anti-idiotype enrichment of producer line, wewill FACS sort the producer line to clone out the brightest 0.1%(following surface staining of producer line with anti-idiotypicantibody). This sorting can be repeated until there is no addedimprovement in mean fluorescence. The producer line is then subclonedusing NK92 as indicator cells, and screened for scFv, SSTR2 (usinganti-HA antibody) or hNIS (using anti-HA or MoAb specific for hNIS) geneexpression by flow cytometry. Subcloning is repeated until a stableclonal producer line is obtained. The most efficient producer clone isselected for cell banking. We plan to use NK92 instead of K562 asindicator line because NK92 is relatively easy to transfect and cloneand has great clinical potential. Previously we used centrifugation toeffect viral attachment and infection of human lymphocytes (see ProgressReport). We plan to further improve the efficiency of retroviralinfection by using fibronectin fragment CH-296 (Takara Shuzo, Otsu,Japan), to augment gene transfer by interaction between VL A-4 onT-lymphocytes and FN adhesion site CS-1,¹³⁶ in conjunction withcentrifugation.^(137,138) In those reports, gene transduction increasedfrom 12% to 18% with centrifugation and to 24% when centrifugation plusfibronectin was used.¹³⁸ The kinetics of surface SSTR2 expression andcytoplasmic HSV1-tk expression will be monitored. Day 40 scFv-modifiedlymphocytes will be analyzed for CD4 CD8, CD56, scFv, SSTR2 (or HSV1-tk)expression. After further expansion in culture, they will be analyzedfor their iodine uptake and efflux. Protein expression can be confirmedby western blot and mRNA by Tagman quantitative RT-PCR¹³⁹ based on theknown genomic/cDNA structure of SSTR2 and the fusion sequence ofscFv-CD28-ζ. Gene copy number based on quantitative PCR method will alsobe used as previously described.¹⁴⁰

Statistical analysis. Data are expressed as the mean ± SEM. Statisticalsignificance of differences is determined by conducting a pairedStudent's t-test.

Results In picking the winner (HSV1-tk, SSTR2, hNIS), the followingcriteria will be used:

-   1. maximal specific activity without damaging cellular function-   2. maximal half-life (retention within cell)-   3. maximal % ID/gm of ex vivo labeled lymphoid cells at 24 and 72    hours in tumor xenograft versus normal organs (liver, spleen and    lung); for NK92 which grows as sc xenograft in SCID mice. Also    considered is maximal % ID/gm of intravenous radiolabeled DOTATOC,    radioiodine or radioiodinated FIAU.

HSV1-tk (˜1 kp) gene transduction using IRES vector and its expressionin human T-cells are now routine.¹⁴¹ Although both hNIS (˜2 kp) andSSTR2 have been transduced by retroviral vector into mammalian cells,efficiency of the IRES construct can vary. It is conceivable that theefficiency of gene transfer and gene expression could also vary betweencloned line NK92 and primary human T lymphocytes. We plan to quantitatethe gene copy number by real time PCR, mRNA by RT-PCR and correlate themwith protein expression by flow cytometry and western blots. In vivobiodistribution of gene-modified NK92 cells and lymphocytes will beverified using immunostaining of mouse tissues and tumors employingbiotinylated anti-idiotypic reagents. Alternatively, quantity of humanlymphocytes in mouse tissues can also be measured by sensitive real timePCR (of transduced genes) as well as RT-PCR for mRNA using mouse ∃-actinand mouse GAPDH, respectively, to calculate relative copy number. Wealso plan to validate in vivo cell-imaging studies using radiolabeledanti-idiotypic reagents. Although anti-idiotypic reagents offer anotheralternative to marker genes for imaging T-cells, these reagents are notwidely available as octreotide (already licensed by the FDA for totalbody imaging) and could be difficult to implement clinically.

In adoptive cell therapies, gene-marking allows precise evaluation ofthe quantity and persistence of these cells in vivo, as well as theirdistribution and function within tissues.¹⁴² In studies of T celltherapy, this is of particular importance since many infused cells willundergo activation-induced death in vivo.¹⁴³ or immune elimination ofgene-modified cells may occur, especially following repeatedinjections.¹⁴⁴ The development of sensitive, accurate and reproduciblemethods to quantify gene-marked cells in peripheral blood and tissuesare essential for defining the long-term fate of transferred cells. Suchpharmacokinetic information is crucial if understanding and optimizationare to be pursued. We want to take advantage of instrumentation andsoftware developed for SPECT and PET/micro-PET imaging. These tools willgive unprecedented precision and dynamic information in future patienttrials.

Specific Aim 2 Pretargeting of CD4+ T-Cells to Improve Adoptive CellTherapy

The fate of CIR gene-modified T-cells in vivo remains unknown in mostcases. Influx of inflammatory cells following local increase in vascularpermeability during complement activation and release of anaphylatoxinsis well known. An active process of recruitment may be equally if notmore important in cellular immunity. The importance of recruitment byCD4+ T cells, chemokines and cytokines, as well as the myriad ofadhesion molecules involved in lymphocyte rolling, adhesion, diapedesis,and movement within the tumor microenvironment have been previouslyemphasized.¹⁴⁵ Distinct roles for Th1 and Th2 for tumor eradication invivo have recently been proposed.¹⁴⁶ While Th1 cells induce a markedlymphocyte infiltration into the tumor mass and eradicate tumor mass viacellular immunity and memory CTL, Th2 cells induce inflammatoryresponses and tumor necrosis through IL-4 recruitment of eosinophils andneutrophils. Th1 cells express high levels of P-selectin, and exhibitstrong LFA-1/ICAM-1 dependent cell-cell interactions and Th2 cellsinteract with extracellular matrix through the integrins. Th1 cells areprobably the lymphocytes responding actively to tumor cells andproducing cytokines, which in turn recruit other effector cellsincluding CD8+ T cells, NKT or NK cells into the tumor tissue. Incontrast, Th2 cells, unable to enter tumor tissue because of theirdefect of adhesion, may accumulate on the endothelium and induce tumornecrosis via TNF-0 and/or release of reactive oxygen intermediates fromeosinophils and macrophages to damage tumor vessels.

T cells homing depends on chemokines and receptors, clearly illustratedin allograft and graft rejection models.¹⁴⁷ CCR4, CXCR3, CCR5, and CCR7are some of the key chemokine receptors for T-cell trafficking. CCR4 isthe major trafficking receptor for systemic memory T cells. A pivotalrole for CCR5 in T-cell migration to tumor sites induced by interleukin12 treatment was recently reported.¹⁴⁸ CXCR3 is present on activatedlymphocytes including CTL and NK cells.¹⁴⁹ Th1 cytokines and CXC3chemokines can direct infiltration of adoptively transferred CD8+ Tcells into the tumor site. CCR7, the lymph node-homing receptor, isexpressed on CD4+ or CD8+ mature T cells. This is important sincemetastatic solid tumors often spread to marrow, bone, lung and liver.CCR7 downregulation may permit these cells to home to nonlymphoidmetastastic sites. T-cells also play an important role in recruitment byusing chemokines. RANTES enhances Th1 and CD8+ CTL responses,¹⁵⁰ whileCTLs can in turn release IL-8, MIP, RANTES, and IP-10.¹⁵¹

General Plan of Work:

Enrichment of CD4+ T-cells. In order to prepare sufficient numbers ofT-cells for in vivo biodistribution studies, cultured CIR-gene modifiedT-cells (>95% scFv positive) will be purified into CD4+ and CD8+populations by affinity chromatography using MiniMac System (Miltenyi).

Homing studies on separated CD4+ and CD8+ populations. Following CIRtransduction and affinity purification, CD4+ cells will be checked byflow cytometry for surface phenotype and cytoplasmic cytokines, plusgene expression by microarrays. In addition, their antigen specificimmune function will be checked in ELISPOT assays (IFN-γ). CD8+ cellswill be analyzed likewise and their cytotoxicity confirmed in ⁵Crrelease assay. The misdistribution of [CIR+imaging]-gene modified CD4+and CD8+ T-cells will be studied in mice with and without tumorxenografts. PET will be used for imaging and quantitative dosimetry. Atspecific time points, mice will undergo necropsy and tissues harvestedfor gamma counting. CD4+ T-cells with CIR but no imaging gene will alsobe tested in the pretargeting model. Indicator cells (both NK92 andT-lymphocytes) carrying the imaging gene, with or without CIR, will betested for their homing response to pretargeted CD4+ T-cells either by(1) radiolabeling in vitro before administration iv, or (2)radiolabeling in vivo after they have had time to home to the tumorsites.

Quantitation by PET will be validated with tissue extraction andanalysis by flow cytometry (quantitation of scFv+ cells). Here, singlecell suspensions will be prepared from tumors and organs (blood, spleen,liver, and lymph nodes) by mechanical disruption and coarse filtering.Live cells will be marked by propidium iodide and their CD3, scFv, CD4and CD8 expression quantified. Tumor cells will be marked with anti-gp58(MoAb 8H9) or anti-GD2 (MoAb 3F8) antibodies. Number of T cells will beexpressed as percent of total cells and per gram tumor or tissue weightfor comparisons. In addition, total DNA and RNA will also be tested forscFv gene copy number and scFv transcript number using PCR and RTPCR,and normalized to mouse ∃-actin and mouse GAPDH, respectively. Againthese will provide independent validation for the quantitationtechniques described above.

Specific Methods:

Phenotypic characterization In addition to gene array analysis(chemokine and receptors, interleukin and receptors) on the CD4+ andCD8+ cells, their surface and cytoplasmic phenotype will also be studiedby FACS analysis at select time points during in vitro culture. Thesemarkers include CD4, CD8, CD25, CD45RO, CD69, VLA-4, LFA-1a, LFA-1-b,L-selectin, CCR4, CCR5, CXCR3, CRTH2, CCR7, cytoplasmic granzyme B,IL-2, IL-4, and IFN-(. Specific antibodies are obtained from the NIHAIDS Resource Program as well from commercial sources: anti-CCR3 (R&Dsystems, Minneapolis, Minn.), anti-CXCR3 (R&D), anti-CCR5 (Pharmingen,San Diego, Calif.), anti-CCR4 (Dr. Chantry, Icos Corporation, Bothell,Wash.) and anti-CCR7 (Pharmingen). Four color fluorescence will beperformed: APC-anti-CD3, PerCP-anti-CD8, FITC-2E9 (anti-idiotype) andPE-antibody (specific for adhesion molecules, cytokines, and chemokinereceptors). Appropriate controls will be included for channelcompensation.

Intracellular cytokine expression For the detection of cytoplasmiccytokines, cells are first cultured in the presence of brefeldin A(Sigma), stained with PerCP-anti-CD4 MoAb, fixed with 4%paraformaldehyde, and treated with permeabilizing solution (50 mM NaCl,5 mM EDTA, 0.02% NaN3, 0.5% Triton X-100, pH 7.5) before staining withPE-conjugated anti-IL4, and FITC conjugated anti-IFN-g for 45 min onice. The percentage of cells expressing cytoplasmic IL-4 (Th2) or IFN-((Th1) is determined by flow cytometry.

Results We test the hypothesis that pretargeting of CD4+ T-cells canincrease the homing efficiency of subsequent injections of NK cells andlymphocytes. Although NK92 is used as an indicator cell in ourexperiments, their broad anti-tumor activity and preliminary clinicalapplications are encouraging evidence of its clinical utility. Since thecurrent limitation of adoptive cell therapy using killer cell lines likeNK92 remains suboptimal in efficiency in tumor targeting, the ability ofCD4+ T-cells to facilitate this tumor-homing property can potentiallyimprove their anti-tumor efficacy, which we will test in xenograftedSCID mice. Our studies will also attempt to characterize the ability ofCD4+ T cells (armed with CIR) to recruit untargeted NK cells anduntargeted T-lymphocytes (i.e. without CIR), and if this recruitmenttranslates into significant anti-tumor effect. It is also possible thatother human white cell populations as well as stem cells can be studiedin this homing model. Although the SCID mice provide a model to testhuman T-cell homing, it has inherent limitations. For example, tumorstroma is mostly mouse-derived, not made up of human stromal cells, andthus are missing certain chemokines and cytokines that can positively ornegatively modulate the T-cell homing properties. In addition, SCIDxenografts are generally made up of mouse-derived vasculature, and assuch will not interact with human lymphocytes or inflammatory cells in aphysiological manner. Nevertheless, these tests will allow us tovalidate the imaging gene technique especially when applied inquantitative PET imaging of gene-modified T-cells. A noninvasivetechnique to quantitate lymphocyte trafficking will undoubtedly requiremuch further refinement, and likely necessitates using human patients.Positive results from these studies will provide the rationale forfurther developments in their clinical translation.

Interpretations and implications The biology of chemokine receptors andadhesion molecules in directing T cell traffic is a rapidly advancingfield. As the science of cytotherapy becomes more sophisticated, purersubpopulations of lymphocytes with defined functions will becomeavailable for in vivo studies. Understanding the cellular cascade inorchestrating tumor targeting will provide crucial information fordiagnostic and therapeutic manipulations. Reliable methods to tracelabel these cells without damaging their cellular function are stilltechnologic gaps in cytotherapy.

Specific Aim 3 Improving Tumor Homing and Tumor Cytotoxicity by usingProfessional T-Lymphocytes (CTL) and NK92 for CIR Gene-Modification

General Plan of Work:

In both specific aims 1 and 2, we propose to use NK92 as our indicatorline. NK91 is a cloned professional NK killer cell line. They havepotential clinical utility in adoptive cell therapy in early humantrials. Antigen specific CTLs, when highly enriched, are also efficientkilling machines ideal for adoptive cell therapy. More importantly,these enrichment steps can also remove substantial numbers ofalloreactive T cells, such that allogeneic CTLs may be safe to use. Thisis particularly relevant when one considers the paucity of healthy Tcells in patients after intensive chemotherapy available forgene-modification. EBV-specific T cells can be selected early after invitro sensitization, while alloreactive T cells are substantiallydepleted by this approach,⁸⁵ enriching the auto/allo ratio by 39-fold.Indeed these EBV-specific T cells can be continually boostedperiodically with EBV-infected cells in vitro, and maintain theirability to home to and kill specifically EBV-lymphomas in Vivo.¹⁴¹ Thespecificity of these CTLs is exquisite, since allogeneic EBV-lymphomasare not killed.

Viral antigen-specific T cells have been successfully used in adoptivetherapies in patients.²⁰⁻²³ Adoptively transferred donor-derivedEBV-specific T cells can effectively eliminate B-cell proliferativedisorders in the post-transplant period: a dramatic proof of principlefor adoptive T-cell approach in cancer therapy, emphasizing theirefficacy and relative safety.^(22,23,25) When genetically tagged withthe neomycin resistance gene using a retroviral vector,²⁶ these CTLs canbe shown to last for up to 18 months.²² The persistence of theseEBV-specific CTLs probably reflects a continual antigenic challenge fromdormant EBV virus residing in the body after primary infection.Recently, Rossig et al have shown that these CTLs can be gene modifiedwith scFv-CIR.¹⁵² We have also demonstrated that complex scFv-CD28-.chimeric gene can be transduced into these CTLs whereupon continualclonal expansion >10⁵ fold for many months (Progress Report).

We propose to arm EBV-specific professional CTLs with scFv-CIR. As CTLsthey are effective and safe, both in vitro and in vivo. They can begene-modified using retrovirus. They mount effective amnanestic CTLresponse. And most importantly, using rapid in vitro selection,⁸⁵alloreactivity can be eliminated. This approach will enable allogeneicCTLs to be used for scFv-based T cell therapy. EBV-specific CTLs fromdesignated normal donors will greatly increase the efficacy and successof gene-modified T cells. More importantly, there is no chance ofcontamination by tumor cells if allogeneic T cells are used.

Specific Methods:

Production and culture of EBV-lymphoblastic cell lines PBMCs at aconcentration of 1×10⁶/mL will be incubated for 24 hours after isolationby Ficoll-Hypaque density centrifugation with the EBV containingsupernatant of the marmoset cell line 95-8 in the presence of 0.5 ugphytohemagglutinin (PHA)-16 (Murex-Diagnostik, Norcross, Ga.) in RPMI1640 (GIBCO, Life Technologies, Grand Island, N.Y.), 10%heat-inactivated fetal calf serum (FCS), 10 U/mL penicillin, 10 ug/mLstreptomycin, and 1% L-glutamine. After 24 hours, cells are washed andrecultured in EBV-containing medium without PHA in 24-well plates at aconcentration of 1×10⁶/mL. Cells are then fed fresh RPMI 1640 with 10%FCS, L-glutamine, penicillin, and streptomycin twice a week and expandedaccording to the growth and cell number. The cells are finallycharacterized by fluorescence-activated cell sorter (FACS) analysisusing CD3, CD 19, and CD20 monoclonal antibodies (Becton Dickinson).Aliquots of the immortalized B-lymphoblastoid cell lines (BLCLs) arefrozen and the remaining cells maintained in culture. Homozygous BLCLsfor the HLA-A and HLA-B alleles, generously provided by Dr. B. Dupont ofMSKCC are maintained in the same medium. PHA blasts are generated byculturing 1×10⁶/mL PBMC with 0.5 ug/mL PHA-16 for 3 days. The cells werewashed and further cultured for 4 days in the presence of 5 IU/mLinterleukin (IL-2) (Collaborative Biomedical Products, Bedford, Mass.).

Generation and culture of EBV-specific CTLs PBMCs are isolated byFicoll-Hypaque density centrifugation of anticoagulated whole blood. Tlymphocytes are positively selected by staining with an anti-CD3phycoerythrin monoclonal antibody (Becton Dickinson) on a MoFlo cellsorter (Cytomation, Fort Collins, Colo.), achieving a purity of morethan 98%. EBV-specific CTLs are generated by stimulating 1×10⁶/mL CD3+cells with 2.5×10⁴/mL autologous BLCLs, which are irradiated with 60 Gyin Iscove's modified Dulbecco's medium supplemented with 10%heat-inactivated human AB serum (Gemini, Calabasas, Calif.), 35-ug/mLtransferrin, 5-ug/mL insulin, 2×10⁻⁵ M ethanolamine, 1 ug/mL palmiticacid, 1 ug/mL linoleic acid, and 1 ug/mL oleic acid (all from Sigma) for6 days in 25-cm² flasks. Cells are then washed, recultured at aconcentration of 1×10⁶/mL, and restimulated with 2×10⁵/mL BLCL at day 7.Cells are either prepared for gene transfer on day 8 (early genetransfer) or kept in culture with restimulations weekly at aneffector-to-target ratio of 5:1. After the third restimulation, T cellsare prepared for gene transfer on day 23 (late gene transfer). A totalof 5 IU of IL-2 (Collaborative Biomedical Products) are added for thefirst time at day 10 to the cultures and 2 to 3 times weekly thereafter.For generation of alloreactive cells, donor T cells are stimulated withfully mismatched allogeneic EBV BLCL. These cells are now routinelygenerated in the Bone Marrow Transplantation Research Laboratory atMSKCC under the supervision of Dr. G. Koehne, co-investigator.

Gene transfer We have shown (see Progress Report) that EBV-specific CTLscan be easily gene-modified using our strategy developed for primaryT-cells. EBV-activated T cells (day 5, day 8, or day 23 of culture) oranti-CD3/anti-CD28-immobilized monoclonal antibody stimulated cells areplaced in fibronectin-coated wells according to the technique describedby Pollok et al¹³⁷ 5 ug/mL of fibronectin fragments (TaKaRa Biomedicals,Shiga, Japan) are coated on nontissue culture treated plates for 2 hoursat room temperature in 6-well plates. Plates were blocked with 1% humanserum albumin for at least 30 minutes and washed twice with PBS. Cellsare plated at a concentration of 10⁶/mL for 24 hours. Viral supernatantis added and spun for 60 min at 1000 rpm at room temperature. Fiftypercent of the supernatant is replaced with fresh medium containing 10%heat-inactivated human AB serum and 10 IU/mL IL-2. Cells will bemaintained in culture at a concentration of 10⁶/mL to 1.5×10⁶/mL.

Flow cytometric analysis Monitoring of the gene expression of scFv of Tlymphocytes will be performed by 2-color flow cytometry using FACScan(Becton Dickinson) by labeling the cells with an anti-scFv-idiotypemonoclonal antibody on ice for 45 minutes. FITC-goat anti-rat antibodyis added for 15 minutes as secondary antibody. After blocking withnormal mouse serum (ICN/ CAPPEL, Aurora, Ohio) for 10 minutes, anti-CD3phycoerythrin (Becton Dickinson) is added for 15 minutes. Cells arewashed twice with PBS after each step and before analysis. Phenotypingof specific CTL lines is performed by gating lymphocytes using forwardsight scatter and sideward sight scatter. Cells are stained withanti-CD3, anti-CD4, and anti-CD8 for T-cell subpopulations. Althoughcells have been purified initially for T lymphocytes, the transducedcells will be reanalyzed for the presence of natural killer (NK) cells,defined as CD3-CD16+CD56+, using anti-CD16 and anti-CD56 monoclonalantibodies (Becton Dickinson).

Cell purification by affinity chromatography Gene-modified cells areprepared for purification using affinity purification on MiniMAC columnsas described in the previous section.

Cytotoxicity assay Cytolytic activity of effector cells is assayedagainst ⁵¹Cr-labeled targets in standard 4-hour release assays. Targetcells include autologous BLCLs, HLA class I mismatched allogeneic BLCLs,and K562 for major histocompatibility complex (MHC)-unrestricted lysisas a parameter for NK cell lysis and PHA blasts. For each donor HLAclass I allele, a BLCL expressing the HLA-A and HLA-B allelehomozygously can be included to determine the HLA restriction of theEBV-specific CTLs. Briefly, 1×10⁶ target cells are incubated with 3700kBq ⁵¹Cr for 1 hour, washed 3 times, and plated in 96 wells.Cytotoxicity is analyzed using 0.8×10⁵ effector cells: 4×10³ targetcells per well in a total volume of 200 uL, at an effector-to-targetratio of 20:1. All targets are plated in triplicate. After an incubationof 4 hours, supernatants are harvested and the specific cytotoxicitydetermined using a microplate scintillation counter (PackardInstruments, Downer's Grove, Ill.). The percentage of specific lysis iscalculated as 100% ×(experimental release—spontaneous release)/(maximumrelease—spontaneous release). Maximum release is obtained by adding 100uL of 5% Triton X-100 to the 100 uL medium containing target cells.Spontaneous release is consistently below 15% of maximum release in allassays.

Comparison of professional killer versus naive T-lymphocyte in tumortargeting and therapy In order to test if CIR-gene modified professionalkiller cells are indeed superior in CIR technology, we compare them withCIR-gene modified naïve T-cells as follows:

-   1. In vitro cytotoxicity (⁵¹Cr release and IFN-(release) against    tumor cell lines-   2. In vivo anti-xenograft activity at various T-cell doses-   3. Quantitative difference in homing measured in % injected dose/gm    over time (for lymphocytes labeled ex vivo or labeled in vivo) by    quantitative PET, tissue counting, immunohistochemistry, and    PCR/RT-PCR-   4. Qualitative difference in CD4+ T-cells in their ability to    recruit either IR-modified T-cells, measured by quantitative PET,    tissue counting, immunohistochemistry, and PCR/RTPCR.

5. Qualitative difference in CD8+ T-cells in their ability to respond topretargeted CD4+ cells, measured by quantitative PET, tissue counting,immunohistochemistry, and PCR/RT-PCR.

Results We expect the use of professional CTLs to greatly increase theefficiency, specificity, and utility of CIR-modified T cells, asrecently reported.152 Since these cells are selected for EBV withminimal alloreactivity the possibility of using healthy tumor-freeallogeneic lymphocytes will increase the chance of full T cell potentialin vivo, a close analogy to adoptive allogeneic T cell therapy ofEBV-lymphoma. Issues in using autologous T cells, such asimmunosuppression by cancer and by chemo-radiotherapy, defective T cellsignaling, and tumor contamination are no longer limiting factors.Clearly, allogeneic cells will be rejected by normal hosts even if theyare HLA-matched. Fortunately, in patients with solid tumors undergoinghigh dose therapy, their immune system is often incapacitated, albeittemporarily. In addition, combination of cyclosporin plus mycophenolatemofetil has been quite effective in suppressing graft versus hostdisease as well as allo-sensitization in preliminary animal studies andearly patient trials.¹⁵³ We expect the immediate post-chemotherapyperiod in patients with solid tumors to be relatively immunosuppressedto allow allogeneic T-cells to survive as least for a brief period ofweeks to exert its antitumor effect. With immunologic recovery, thesegene-modified cells will be eliminated, together with the risk of thembecoming cancerous or causing long term autoimmunity. Autologous T cellswould have recovered enough by then to allow autologous EBV-specificCTLs to be used. Whether committed EBV-CTLs have shorter life spans thannaive T cells after CIR gene-modification remain to be determined. It islikely to expect these preprogrammed professional killer cells(EBV-specific CTL), being preselected in their priming period to expressthe appropriate repertoire of adhesion molecules (ICAM and selecting),cytokine/interleukins (e.g. IL2, IL4, IL12) and receptors (IL2R, IL7R,IL15R), chemokines (RANTES, IP10) and receptors (CCR5, CXCR3, CCR4), toshow better anti-tumor activity than primary T-cells.

Interpretations and Implications The identification of an optimal genedesign in the allogeneic setting will increase the likelihood ofclinical benefit of CIR technology. The ability to produce large clonalpopulations of tumor specific T-cells from normal donors for lymphocytetherapy will increase the chance of its successful clinical translation.

TABLE 4 NK92 surface phenotype by FACS NK Phenotype CD56 +++ CD16 − CD3− CD4 − CD8 − CD2 ++ CD7 +++ CD25 (IL2Ra) + CD122 (IL-2Rb) ++ Celladhesion molecules (CAM) Ig superfamily CD54 (ICAM-1) +++ CD102 (ICAM-2)++ CD50 (ICAM-3) + Integrins B1 CD29 (B1 integrin B ++ chain) CD49d(VLA-4 a chain) +++ CD49e (VLA-5 a chain) − B2 CD18 (B2 integrin B +++chain) CD11a (LFA-1 a chain) +++ CD11b (Mac-1 a chain) − CD11c (p150/95a + chain) Other CAM CD44H +++ CD44R1 ++ CD58 (LFA-3) +++ NKregulatoryReceptors CD158a − CD158b − KIR70 − CD94 ++ NKG2A +++ Miscellaneous CD28++ CD152 (CTLA-4) − CD80 + CD86 ++ C95 (Fas) + FasL −/+ CD69 ++ CD34 −CD43 +++ CD48 +++

TABLE 5 8H9-scFv- CD28-zeta T- NK9 cells 2 IL1A — — IL1B W — FIL1 (ε) WW FIL1 — — FIL1 (ζ) Y Y IL-1H1 — — IL2 — — IL3 W W IL4 W — IL5 W — IL6 YW IL7 W Y IL8 — W IL9 Y Y IL10 — W IL11 Y Y IL12A Y W IL12B — — IL13 W —IL14 W W IL15 Y Y IL16 W W IL17 W W IL17B W W IL17C — W IL17E Y Y IL17FY Y IL18 — — IL19 Y Y IL20 — — IL21 Y Y IL22 Y Y IL23A Y Y IL24 W W IL26— —

TABLE 6 8H9-scFv- CD28-zeta T- NK9 cells 2 IL1R1 W W IL1R2 W Y IL1RL1 —— IL1RL2 — — IL1RAP W W IL1RAPL1 W W IL1RAPL2 — W IL1RN W — IL1HY1 — —IL2RA W W IL2RB W W IL2RG Y Y IL3RA — — IL4R — — IL5RA — — IL6R — WIL6ST — — IL7R — — IL8RA — — IL8RB — — IL9R W W IL10RA Y Y IL10RB W WIL11RA W W IL12RB1 W W IL12RB2 — W IL13RA1 W — IL13RA2 W — IL15RA W WIL17R Y Y IL18R1 — — IL18BP W Y IL18RAP — — IL20RA W W IL21R Y Y IL22R WY IL22RA2 Y Y

TABLE 7 8H9-scFv-CD28- zeta T cells NK92 ENA-78 W — Eotaxin W — GCP-2 YY I-TAC (IP9) — — (SCYB11) lymphotactin Y Y MCP-1 (SCYA2) — — MCP-2 — —MCP-3 — — MCP-4 — — MDC Y Y MIP-1 delta W Y MIP-1a Y Y MIP-1b Y Y MIP-2(SCYA21) W W MIP-3a — — MPIF-1 Y Y MPIF-2 — — P10 (IP 10) — — PARC W YSCYA19 Y Y SCYA5 (RANTES) Y Y SCYB13 Y Y SCYC2 Y Y SCYE1 W Y SDF1 Y YSDF2 Y Y TARC (SCYA17) Y Y

TABLE 8 8H9-scFv-CD28- zeta T-cells NK92 CCR1 W W CCR2 Y Y CCR3 — — CCR4Y Y CCR5 Y Y CCR6 W — CCR7 W W CCR8 W — CCR9 W W CX3CR1 — — CXCR4 W YCXCR5 (BLR1) — — XCR1 Y Y

TABLE 9 IC50 values (nM) for Octreotide analogs against ¹²⁵I-SST-14 SSTRPeptide 1 2 3 4 5 Octreotide >1000 2 187 >1000 22 DTPA-Octreotide >100012 387 >1000 299 DOTA-Octreotide >1000 14 27 >1000 103DOTA-Tyr3-Octreotide >1000 14 880 >1000 393 Y-DOTA-Tyr3-Octreotide >100011 389 >1000 114 Ga-DOTA-Tyr3-Octreotide >1000 2.5 613 >1000 73In-DTPA-Octreotide >1000 22 182 >1000 237 Y-DOTALAN >1000 23 290 >100016 Re0-P829 >1000 2.5 1.5 >1000 2

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1. A method for treating a mammalian subject having a malignant tumorcomprising cells expressing a target antigen, the method comprising,contacting a population of cells comprising natural killer cells or Tcells with a polynucleotide encoding a chimeric scFv (single chainantibody) directed against a target antigen gp58 which is recognized byantibody 8H9, wherein the chimeric scFv comprises heavy and light chainvariable regions from the 8H9 antibody, a transmembrane domain and asignaling domain; culturing the cells under conditions suitable forexpressing the chimeric scFv-encoding polynucleotide; contacting cellsexpressing the chimeric scFV-encoding polynucleotide with ananti-idiotype directed against the chimeric scFv under conditionssuitable for binding of the anti-idiotype to the chimericscFv-expressing cell, thereby stimulating the formation and growth ofone or more chimeric scFv-expressing clones; selecting one or more ofthe chimeric scFv-expressing clones; and introducing the one or morechimeric scFv-expressing clones into the mammalian subject.
 2. Themethod of claim 1, wherein the malignant tumor is a human malignanttumor.
 3. The method of claim 2, wherein the human malignant tumor is abrain tumor, a sarcoma or a neuroblastoma.
 4. The method of claim 2,wherein the human malignant tumor is a neuroblastoma, a mixed glioma, aoligodendroglioma, astrocytoma, a meningioma, a schwannoma, amedulloblastoma, a neurofibroma, a neuroglial tumor, a ependymoma or apinealblastoma.
 5. The method of claim 2, wherein the human malignanttumor is Ewing's primitive neuroectodermal tumor, a rhabdomyosarcoma, aosteosarcomas, a desmoplastic small round cell tumor, a synovialsarcomas, a leiomyosarcoma, or a malignant fibrous histiocytoma.
 6. Themethod of claim 2, wherein the human malignant tumor is a melanoma, ahepatoblastoma, Wilm's tumor, a rhabdoid tumor or an adenocarcinoma. 7.The method of claim 1, wherein the transmembrane and signaling domainsare the T cell receptor transmembrane and signaling domains.
 8. Themethod of claim 1, wherein the signaling domain comprises a zeta chainand optionally, a TCR-70 zeta-associated protein.
 9. The method of claim1, wherein the transmembrane and signaling domains are from CD28.